All in the family: the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases

Kelly G. Ten Hagen, Timothy A. Fritz and Lawrence A. Tabak1

Biological Chemistry Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Md 20892, USA

accepted on September 6, 2002

Abstract

Mucin-type linkages (GalNAc{alpha}1-O-Ser/Thr) are initiated by a family of glycosyltransferases known as the UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (ppGaNTases, EC 2.4.1.41). These enzymes transfer GalNAc from the sugar donor UDP-GalNAc to serine and threonine residues, forming an alpha anomeric linkage. Despite the seeming simplicity of ppGaNTase catalytic function, it is estimated on the basis of in silico analysis that there are 24 unique ppGaNTase human genes. ppGaNTase isoforms display tissue-specific expression in adult mammals as well as unique spatial and temporal patterns of expression during murine development. In vitro assays suggest that a subset of the ppGaNTases have overlapping substrate specificities, but at least two ppGaNTases (ppGaNTase-T7 and -T9 [now designated -T10]) appear to require the prior addition of GalNAc to a synthetic peptide before they can catalyze sugar transfer to this substrate. Site-specific O-glycosylation by several ppGaNTases is influenced by the position and structure of previously added O-glycans. Collectively, these observations argue in favor of a hierarchical addition of core GalNAc residues to the apomucin. Various forms of O-glycan pathobiology may be reexamined in light of the existence of an extensive ppGaNTase family of enzymes. Recent work has demonstrated that at least one ppGaNTase isoform is required for normal development in Drosophila melanogaster. Structural insights will no doubt lead to the development of isoform-specific inhibitors. Such tools will prove valuable to furthering our understanding of the functional roles played by O-glycans.

Key words: O-glycosylation / O-linked oligosaccharides / UDP GalNAc:polypeptide N-acetylgalactosaminyl-transferases / UDP GalNAc

Introduction

As we enter the postgenomic era, systematic functional analysis of co- and posttranslational modifications, such as glycosylation, will become increasingly important. Because carbohydrates cannot be "mutated" directly, considerable effort has been made to characterize the enzymatic machinery responsible for the synthesis of complex carbohydrates. Mutation of the ion pumps (e.g., Dürr et al., 1998Go), sugar-nucleotide transporters (e.g., Oelmann et al., 2001Go), and glycosyltransferases (Marth, 1996Go; Muramatsu, 2000Go; Furukawa et al., 2001Go) responsible for assembling an oligosaccharide within the endoplasmic reticulum (ER) and Golgi complex will allow for powerful (albeit indirect) tests of the biological function of the carbohydrate side chains. With the application of molecular cloning, it is apparent that the "one-enzyme: one-linkage" concept (Roseman, 1970Go) is not universal and that some glycans may be assembled by the action of any one of a number of highly related transferases. Thus, each family member must be identified and characterized before a clear delineation of the biological function of each glycosyltransferase can be established.

Mucin-type linkages (GalNAc{alpha}1-O-Ser/Thr) are initiated by a family of glycosyltransferases called the UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (ppGaNTases, EC 2.4.1.41). These enzymes transfer GalNAc from the sugar donor UDP-GalNAc to serine and threonine residues. They belong to family 27 of retaining nucleotide-diphospho-sugar transferases (Campbell et al., 1997Go, 1998Go) based on amino acid sequence similarities (see Coutinho and Henrissat, 1999Go). Because O-linked glycosylation proceeds step-wise (Strous, 1979Go), addition of GalNAc to serine or threonine represents the first committed step in mucin biosynthesis. Despite this seeming simplicity, multiple ppGaNTase family members appear to be necessary to fully glycosylate their protein substrates.

O-glycans impart unique structural features to mucin glycoproteins and numerous membrane receptors (Jentoft, 1990Go; Moody et al., 2001Go; Xu and Weiss, 2002Go). Structure–function studies have demonstrated that O-glycans also impart resistance to thermal change and proteolytic attack in a number of diverse proteins (e.g., glucoamylase [Sauer et al., 2000Go]; apolipoprotein A [Garner et al., 2001Go]). O-linked carbohydrate side chains function as ligands for receptors (e.g., in host–microbial interactions [Hooper and Gordon, 2001Go]; lymphocyte and leukocyte homing [Yeh et al., 2001Go; Somers et al., 2000Go]) and as signals for protein sorting (Alfalah et al., 1999Go; Altschuler et al., 2000Go; Breuza et al., 2002Go; Naim et al., 1999Go; Zheng and Sadler, 2002Go).

ppGaNTase activity was first reported some 35 years ago by McGuire and Roseman (1967)Go, who used a microsomal fraction derived from ovine submandibular glands as the enzyme source and deglycosylated ovine submandibular gland mucin as the substrate. Interestingly, the authors noted, "The remarkable degree of specificity exhibited by the transferase towards OSP (the deglycosylated mucin used as the substrate) was unexpected in view of the large number of serine and threonine residues found in the molecule and the fact that many of the inactive substrates also contained these hydroxyamino acids." Thus, it was observed that not all hydroxyamino acids acquire GalNAc and therefore some rule(s) must exist to discriminate the subset of serines and threonines that do become modified.

How many ppGaNTases are there (or, What's in a name)?

A ppGaNTase was purified from an ascites hepatoma (Sugiura et al., 1982Go); it had an apparent molecular mass of 55,000 Da. Elhammer and Kornfeld (1986)Go subsequently reported purification of a ppGaNTase from bovine colostrum and a murine lymphoma, which had an apparent molecular mass of 70,000 Da. Although it was not appreciated fully at the time, this may have represented the first evidence for a family of closely related ppGaNTase isoforms. Distinct ppGaNTase activities were resolved from extracts of porcine and ovine submandibular glands by the Clausen group (Sørensen et al., 1995Go); they found that these distinct activities (named ppGaNTase-T1 and -T2) were differentially expressed in tissues surveyed. This finding underscores the difficulty in resolving the signals that direct acquisition of O-glycans using tissue or cell extracts as the enzyme source.

Molecular cloning has allowed for the unambiguous identification of distinct ppGaNTases. To date, 12 mammalian members of the ppGaNTase enzyme family have been cloned and functionally expressed from various species; each isoform has been numbered in order of its cloning and functional expression (Table I). An analysis in silico, comparing the most recent annotated public (NCBI human genome gscan_mRNA database) and private (Celera Human Genes database) human genome databases and Ensembl databases, suggests that there are as many as 24 human ppGaNTase genes. Parenthetically, neither database has the complete complement of isoforms cataloged as yet (see Hogenesch et al., 2001Go for general commentary about the relative overlap between the public and private genome databases).


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Table I. Functional ppGaNTases characterized to date

 
Many years ago, Ephraim Racker admonished that one should not "waste clean thinking on dirty enzymes" (in Kornberg, 1991Go). Given the robustness of current genomic and EST databases, we feel that a useful corollary is that "it's not an enzyme until you prove it's an enzyme." Indeed, there is at least one report of a ppGaNTase pseudogene in the literature (Meurer et al., 1996Go). Additional putative ppGaNTase isoforms have been reported in the literature, but functional activities have not yet been demonstrated (e.g., White et al., 2000Go).

To avoid further confusion, we propose that in the future, numbers be used only after evidence for enzymatic activity is obtained (Table II). Putative isoforms could be assigned letters, pending confirmation of enzymatic activity. Because ppGaNTase-"T8" already appears in the public databases, we reserve "-T8" for this isoform (with the expectation that it will prove to be a functional enzyme). Similarly, because the presence of two "-T9's" in the database is confusing, we propose to name the ppGaNTase-T9 described by Ten Hagen et al. (2001)Go, ppGaNTase-T10 and reserve "-T9" for the isoform described by Toba et al. (2000)Go that has recently been shown to have enzymatic activity (Zheng et al., 2002Go). Following this proposed scheme, the next isoform with demonstrated (published) activity should be assigned ppGaNTase-T11 (Schwientek et al., 2002Go), then -T12 (Guo et al., 2002Go) followed by -T13 (Zheng et al., 2002Go), and so on.


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Table II. Proposed nomenclature for ppGaNTases

 
Enzymatic mechanism and structural features

Conceptual translation of ppGaNTase cDNAs reveals that these enzymes are type II membrane proteins, characterized by a short (4–24 aa) N-terminal cytoplasmic tail, followed by a small (15–25 aa) transmembrane anchor, which is tethered to a large (>450 aa) segment in the lumen of the Golgi by a stem region of variable length. The function of the N-terminal cytoplasmic tail in ppGaNTases has not yet been determined. The transmembrane domain has been implicated in the localization of glycosyltransferases (Opat et al., 2001Go), but this has not yet been established for any of the ppGaNTases as yet. ppGaNTase-T5 (Ten Hagen et al., 1998Go) has the longest stem region of any glycosyltransferase characterized thus far (~400 aa), containing seven potential N-linked glycosylation sites. It has been suggested that this region serves to extend the catalytic domain into the lumen of the Golgi and/or mediate interaction with other proteins within the Golgi complex (Ten Hagen et al., 1998Go; Breton et al., 2001Go).

Because ppGaNTases are found in external secretions such as colostrum (e.g., Hagen et al., 1993Go), it is likely that the stem region is susceptible to proteolytic cleavage via a membrane protein secretase (Hooper et al., 1997Go). For example, ß-site amyloid precursor protein cleaving enzyme 1 has recently been identified as a membrane-bound aspartic protease that cleaves the sialyltransferase responsible for producing sialic acid {alpha}2,6 galactose linkages (Kitazume et al., 2001Go). This represents another potential layer of control of the activity of members of this family in that up-regulation of specific proteases could liberate specific ppGaNTases into the extracellular compartment of the secretory pathway of cells.

Wragg et al. (1997)Go demonstrated that diethylpyrocarbonate-mediated inactivation of ppGaNTase-T1 was enhanced by the presence of UDP-GalNAc, suggesting that binding of the sugar donor induces a conformational change in the enzyme. However, the only kinetic data available for ppGaNTases thus far (Wragg et al., 1995Go) indicates that the reaction proceeds via a random ordered sequential mechanism. This is in contrast to work on other retaining glycosyltransferases. Kinetic studies of LgtC from Neisseria meningitidis revealed that UDP-Gal binds to the glycosyltransferase first, followed by lactose in an ordered sequential mechanism (Persson et al., 2001Go). Similarly, structural studies of UDP-Gal:ß-galactoside {alpha}1,3 galactosyltransferase (Gastinel et al., 2001Go; Boix et al., 2001Go) have suggested a donor-induced conformational change in the enzyme, although it has not yet been determined if substrate binding is random or ordered (Zhang et al., 2001Go).

Glycosyltransferases that retain the anomeric configuration of the sugar nucleotide bond, by analogy to retaining glycosidases, are thought to work via a double displacement mechanism (Bourne and Henrissat, 2001Go). A nucleophile would be predicted to lie close to the ß face of UDP-GalNAc. Aspartic or glutamic acids are typical nucleophiles, although it has been pointed out that the carbonyl oxygen in an amide side chain could play this role as well (Bourne and Henrissat, 2001Go). Either histidine or carboxylate acid–containing amino acids would be expected to coordinate the preferred Mn2+ cofactor, which is proposed to assist in the binding of UDP-GalNAc. However, to date, there is no evidence for the covalent intermediate that would be required (Davies, 2001Go). One possible approach would be to trap the intermediate using a fluorinated derivative of UDP GalNAc (i.e., 5-deoxy-5-[19]-Fluoro-GalNAc) (see Withers et al., 1990Go). By using the heavy form of F one could also confirm the inversion of the anomeric configuration that is predicted to occur with the intermediate. Unfortunately, the synthesis of 5-deoxy-5-[19]-Fluoro-GalNAc has not yet been achieved.

No crystal structure has yet been solved for the ppGaNTases, although a number of groups are reportedly working toward this goal. In the interim, sequence analysis and computer modeling have been employed to gain insight into the structural features that underlie the enzymatic activities and specificities of these enzymes. Using a combined bioinformatics/site-directed mutagenesis approach, Hagen et al. (1999)Go have proposed that ppGaNTases share a common structural fold containing a parallel ß-sheet flanked by {alpha}-helices. Three distinct domains were identified (Figure 1). The first domain, consisting of 112 aa, comprises the glycosyltransferase motif 1 (GT1) and represents the first half of the catalytic unit. Within this domain is the D209 X H211 ("D X D"; see Wiggins and Munro, 1998Go) sequence that by homology with glycosyltransferases whose crystal structures have been determined, would appear to be responsible for Mn2+ coordination as well as binding both GalNAc and ribose of the sugar nucleotide donor (Unligil et al., 2000Go). Also included within this domain are residues E127 and D156 that are invariant among all forms of ppGaNTases studied to date. Mutagenesis of either residue to Q resulted in the loss of >98.8% activity relative to the wild-type enzyme in ppGaNTase-T1. Cys212 and Cys214 are conserved in ppGaNTase-T1, -T2, -T3, -T4, and -T6; in ppGaNTase-T1 these two cysteine residues are free and appear to contribute to UDP-GalNAc binding, presumably via hydrogen bonding with UDP (Tenno et al., 2002Go).



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Fig. 1. Domain structure of the ppGaNTase family. (A) Consensus sequence diagram of several ppGaNTases with demonstrated activity. Regions in white, pink, red, and black represent, respectively, 0–29%, 30–69%, 70–99%, and 100% sequence identity. The positions of the transmembrane (TM), stem, catalytic (glycosyltransferase 2 domain, GT1, and Gal/GalNAc T motifs) and ricin domains are indicated by the colored arrows. The glycosyltransferase 2 domain was identified from the Pfam database, the ricin domain was identified from the SMART (Simple Modular Architecture Research Tool) database, and the GT1 and Gal/GalNAc T motifs were described in Hagen et al. (1999)Go. The asterisk shows the location of the conserved DXH motif likely to be involved in sugar nucleotide binding. (B) ClustalX alignment of the catalytic domains of the transferases where the colored arrows correspond to the same residues as A. Single-letter amino acid abbreviations are shown for strictly conserved residues. A and B were created using sequences of mouse ppGaNTases 1–4, 7, and 11; rat ppGaNTases 5 (stem region removed) and 10; and human ppGaNTase 6.

 
The second domain consists of 41 aa and contains the D310XXXXXWGGENXE322 sequence. The subsequence WGGE(D) contains the catalytic general base of the ß1,4-galactosyltransferase family (Gastinel et al., 1999Go). Mutation of D310, G319, or G322 within this second domain results in >98% loss of activity relative to wild-type enzyme. The C-termini of virtually all known ppGaNTases end with a ricin-like motif (Hazes, 1996Go; Imberty et al., 1997Go); the known exception is gly-8 from Caenorhabditis elegans. However this form has not yet been demonstrated to have catalytic activity (Hagen and Nehrke, 1998Go). Point mutations within this region in mouse ppGaNTase-T1 do not compromise enzymatic activity in vitro (Hagen et al., 1999Go). However, Hassan et al. (2000)Go presented evidence that a single amino acid substitution within the putative lectin-like domain of ppGaNTase-T4 compromises its ability to glycosylate a glycopeptide substrate but leaves essentially unaffected the activity toward two nonglycosylated peptides. This suggests that the lectin-like domain or at least the single residue mutated in the ppGaNTase-T4 isoform may be involved in the glycopeptide transferase activity observed.

Structure–function insights will allow for rational modeling of global and isoform-specific inhibitors of the ppGaNTases. Such tools will prove valuable to furthering our understanding of the functional roles played by O-glycans. Wragg et al. (1995)Go reported that the dead-end peptide analog PPDAAGAAPLR acts as a competitive inhibitor of the EPO-T peptide (PPDAATAAPLR) for ppGaNTase-T1. Thus, neither serine nor threonine are required for binding to ppGaNTase-T1. Most recently, the Bertozzi group (Yu et al., 2002) has identified two small molecules that inhibit ppGaNTase-T1 (Ki = 8 µM), (-T2, -T3, -T4, -T5, -T7, and -T10) by screening a 1338-member library of uridine analogs that had previously been used to identify an inhibitor of UDP-4-epimerase (Winans and Bertozzi, 2002Go). The availability of small molecule inhibitors of O-glycosylation may facilitate efforts to crystallize this class of glycosyltransferases as well as provide an important tool to study the biological functions associated with O-glycans.

Evolutionary history

In evolutionary terms, the ppGaNTase family is quite old. Although no evidence for O-linked GalNAc is found in Saccharomyces cerevisae, the occurrence of a Galß1, 3GalNAc-Ser/Thr linkage in the fungus Cordyceps ophioglossoides has been reported (Kawaguchi et al., 1986Go). Significantly, the ppGaNTase family has been identified both in silico and biochemically in both C. elegans (Hagen et al., 2001Go; Hagen and Nehrke, 1998Go) and Drosophila (Ten Hagen and Tran, 2002Go; Schwientek et al., 2002Go).

Mika et al. (2001)Go recently analyzed phylogenetic trees for a number of glycosyltransferase families. They estimate a divergence of ß1,4galactosyltransferases from ppGaNTases some 1200–1500 million years ago (MYA) corresponding to the development of metazoans from the early eukaryotes. In contrast to other glycosyltransferase families in which gene duplication occurs during the early period of vertebrate lineage development, ppGaNTases underwent gene duplication very early—between 800 and 1200 MYA, prior to the development of deuterostomes from the metazoans. The evolutionary rates of each glycosyltransferase were estimated by calculating the numbers of synonymous and nonsynonymous nucleotide substitutions between human and rodent glycosyltransferase genes. ppGaNTase-T7 displays the lowest rate of evolution among the 55 glycosyltransferases studied. ppGaNTase-T5 had the highest rate of evolution among the ppGaNTases and the ninth fastest overall rate among all transferases studied.

Hagen and colleagues (2001)Go have pointed out that sequence divergence among any of the different C. elegans ppGaNTase isoforms is greater than that of many of the nematode/mammalian (putative) orthologs. Sequence and phylogenetic analysis has indicated highest similarity among ppGaNTase-T3 and -T6, -T4 and -T12, and -T8 and -T9 (Bennett et al., 1999aGo; Schwientek et al., 2002Go; Guo et al., 2002Go). Interestingly, Drosophila T1 (Drosophila pgant35A; Ten Hagen and Tran, 2002Go) and ppGaNTase-T11 do indeed share both phylogenetic and catalytic similarity. Preliminary evidence was also presented for similarity between Drosophila T2 and ppGaNTase-T7 (Schwientek et al., 2002Go). Collectively, these observations argue that the ppGaNTases are responsible for biologically significant functions that have been conserved during evolution.

Why does the ppGaNTase family need to be so large?

Differential expression
Transcripts encoding a number of ppGaNTase isoforms have been localized in staged mouse embryos, demonstrating that there are differences in their spatial and temporal patterns of expression during development (Figure 2; see Kingsley et al., 2000Go for additional detail). ppGaNTase-T1, -T2, -T4, and -T9 (now designated -T10) were expressed more ubiquitously than ppGaNTase-T3, -T5, and -T7. Organ systems with discrete accumulation patterns of ppGaNTase family members include the gastrointestinal tract (intestine, liver, stomach, submandibular gland), nervous system (brain, eye), lung, bone, yolk sac, and the developing craniofacial region. The pattern in the craniofacial region included differential expression by family members in developing mandible, teeth, and tongue and in discrete regions of the brain, including the pons and migratory differentiating neurons. ppGaNTase-T1 accumulates at high levels in the subset of neural crest that form the mandibular bone. ppGaNTase-T5 accumulates in a very specific subset of cells at the most ventral portions of the E12.5 maxilla and mandible. At E14.5, ppGaNTase-T9 (now -T10) transcripts localize to the neural crest that contributes to the formation of the mandibular bone and teeth. The unique spatiotemporal expression of the different ppGaNTase family members during development suggests unique roles for these products.



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Fig. 2. Overview of expression of ppGaNTase family members during development. mRNA accumulation patterns for seven ppGaNTase family members (-T9 [now designated -T10], -T1, -T2, -T3, -T4, -T5, and -T7, indicated at left) are presented at four times during embryonic development. Column A shows E7.5, late streak to early neural plate stage, Theiler stage 10–11; column B shows E12.5, Theiler stage 21; column C shows E14.5, Theiler stage 22–23; and column D shows E 16.5, Theiler stage 25. Message intensity (red) is overlaid on tissue (blue). The size bar in column A represents 100 µM, in B represents 250 µM, and in C and D represents 1000 µM. BF, brightfield; ys, yolk sac; ep, embryo proper; m, mandible; li, liver; lu, lung; I, intestine; sm, submandibular gland; e, eye. Reprinted with permission, from Kingsley et al. (2000)Go.

 
Each ppGaNTase displays a unique pattern of transcript expression across tissues derived from adult rodents or humans as ascertained by northern blot analysis or real-time polymerase chain reaction (summarized in Table I). Although some isoforms are expressed specifically in a subset of tissues (ppGaNTase-T5, -T7, -T10, -T11, and -T12), others are more ubiquitously expressed (ppGaNTase-T1 and -T2). ppGaNTase-T1 and -T2 are detected in connective tissue potentially contributing to the signal detected from some tissues and organs (Mandel et al., 1999Go).

The Clausen group has prepared a series of monoclonal antibodies against human ppGaNTase-T1, -T2, -T3, -T4, -T6, and -T11 (Bennett et al., 1998Go, 1999aGo; Mandel et al., 1999Go; Schwientek et al., 2002Go). These reagents could prove valuable in delineating patterns of isoform-specific expression in tissues, organs, and cells. For example, Schwientek et al. (2002)Go recently employed these reagents to demonstrate differential expression of ppGaNTases in human kidney. Unfortunately, few studies have been published with other normal tissues and organs (summarized in Table III).


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Table III. Immunolocalization of ppGaNTases

 
Several studies have examined ppGaNTase expression in cancer cell lines and malignant tissues. Steady-state levels of ppGaNTase-T3 mRNA were variable among the cells examined with elevated levels of expression observed from cell lines derived from moderately to well-differentiated pancreatic adenocarcinomas (Sutherlin et al., 1997Go). Nomoto et al. (1999)Go, using a polyclonal antibody to ppGaNTase-T3, demonstrated that this isoform was expressed in adenocarcinoma cell lines but not in other carcinoma cell lines. Most recently, Shibao and colleagues (2002)Go concluded that expression of ppGaNTase-T3 is a useful indicator of prognosis in patients with colorectal carcinoma. Strong expression of ppGaNTase-T3 was associated with a strong likelihood of survival. It is not known if ppGaNTase activity is functionally related to reduced invasiveness observed for tumors expressing high levels of this isoform.

Enzyme activity and specificity
Only a subset of hydroxyamino acids is substituted with GalNAc in the mammalian proteome (Apweiler et al., 1999Go). Thus, rules must exist that specify which serines and threonines will become decorated with O-glycans. Despite intense investigation, no consensus sequon has emerged that is both necessary and sufficient for O-glycosylation to occur. Although most work has concentrated on primary sequence context, several studies have attempted to address the influence of higher-order structure on the acquisition of O-glycans.

In vitro assays with synthetic peptide substrates and cell/tissue extracts as the enzyme source. Studies carried out over four decades have employed synthetic peptide substrates to gain insight into ppGaNTase activity. The earliest surveys examined the glycosylation of the tetrapeptide TPPP (derived from the bovine myelin protein sequence) and variants using tissue extracts as an enzyme source (Young et al., 1979Go; Hughes et al., 1988Go; Briand et al., 1981Go; Cottrell et al., 1992Go). The octapeptide VTPRTPPP was determined to be the best of these substrates, and evidence suggested that both threonine residues were glycosylated (Cottrell et al., 1992Go). Additional studies have been conducted with tissue extracts serving as an enzyme source in which peptides based on the tandem repeating structures of the membrane-bound mucin MUC1 (Nishimori et al., 1994aGo,bGo; Stadie et al., 1995Go), MUC2 (Tetaert et al., 1994Go; Inoue et al., 1998Go, 2001Go; Iida et al., 2000Go; Kato et al., 2001aGo,bGo), and the respiratory mucin MUC5AC (Hennebicq et al., 1998aGo,bGo) have been used as substrates. Collectively, these studies emphasize the potential role of primary amino acid sequence in signaling for sites of O-glycosylation and the preferential acquisition of O-linked GalNAc by threonine versus serine residues.

Assays conducted with reporter peptide or protein substrates transfected into different cell backgrounds. Nehrke et al. (1996Go, 1997Go) have examined the glycosylation of single-site reporter peptide substrates in different cell backgrounds. A broad range of substrates is tolerated in different cell backgrounds without any untoward effect on O-glycan acquisition. De Haan et al. (1998)Go examined the requirements for O-glycosylation of the mouse hepatitis virus membrane protein and reported similar findings. However, the presence of charged amino acids at positions -1 and +3 (relative to the O-glycosylation site) markedly diminishes the acquisition of sugar (Nehrke et al., 1996Go, 1997Go). Charge density (i.e., overall number of charged residues) does not seem to play a significant role because acidic residues at positions -2, +1, and +2 had no inhibitory effect. The presence of charged residues at positions -1 and +3 do not have any effect on the predicted secondary structure of the substrates. In a normal helical structure the substituents on the amino acids at the -1 and +3 positions lie in the same outwardly facing plane. Hence, charged residues at these sites may impede the entry point of the ppGaNTase for peptide substrate, thus explaining why glycosylation is inhibited although there is no impact on the secondary structure of the substrate per se. However, when cells were maintained at a lower temperature (23°C), glycosylation of peptides containing a charged residue at position -1 was restored to wild-type levels. Moreover, the glycosylation of double mutants at positions -1 and +3 (i.e., substituting a charged residue) was also enhanced when cells were incubated at 23°C (Nehrke et al., 1996Go). One possible explanation is that a novel ppGaNTase is induced by the temperature shift (see Henle et al., 1990Go). Alternatively, the increase in transit time of the secreted reporter substrate may enhance Golgi "contact time," allowing for a more complete glycosylation to occur.

Müller and Hanisch (2002)Go have recently estimated the density of O-glycosylation for a truncated (containing six tandem repeats) MUC1 reporter expressed in four breast cancer cell lines. In three of the cell lines (T47D, ZR75-1, and MDA-MB231) the recombinant reporter was substituted with three to five GalNAc residues with the pentaglycopeptide being the predominant species. In the fourth cell line (MCF-7) the recombinant reporter was substituted with two to five GalNAc residues, but the predominant form was the triglycopeptide, indicating a lower O-glycosylation density. One explanation for these findings is that MCF-7 cells lack the complement of ppGaNTases required to achieve the density observed in the other three cell lines.

Database approaches. The validity of database approaches is dependent on the accuracy of O-glycan assignment. Relatively few O-glycan sites have been mapped unambiguously and the majority of these are single sites (for a brief review, see Gooley and Williams, 1994Go). Recent technical advances in mapping O-glycosylation, including the use of Edman degradation (Gooley and Williams, 1994Go; Gerken et al., 1997Go) and mass spectrophotometric methods following various modes of chemical derivatization (for review, see Hanisch et al., 2001aGo; Mirgorodskaya et al., 2001Go; Czeszak et al., 2002Go, and references therein), have made it possible to map native multisite substrates.

The original analyses of O'Connell et al. (1991)Go and Wilson et al. (1991)Go comparing the nature of amino acid sequences flanking hydroxyamino acids that are or are not O-glycosylated was recently updated by Christlet and Veluraja (2001)Go. The principal findings remain unchanged. There is a high frequency of proline, serine, threonine, and alanine residues flanking O-glycosylation sites. In particular, proline is most often found at positions -1 and/or +3 relative to single glycosylation sites. In contrast to single sites of O-glycosylation where acidic residues were not found with great frequency (O'Connell et al., 1991Go; Wilson et al., 1991Go), aspartic acid and glutamic acid are found often around multiple glycosylation sites (Christlet and Veluraja, 2001Go). Elhammer et al. (1993)Go used a database approach to suggest that ppGaNTases have an active site that accommodates eight aa residues, which is in close approximation to what was subsequently reported by Nishimori and colleagues (1994aGo,bGo).

Several neural network approaches have been explored to predict O-glycosylation sites (Cai et al., 1997Go, 2002Go; Hansen et al., 1998Go). NetOGlyc employs a jury of artificial neural networks to recognize sequence context and surface accessibility and is available at the Web site www.cbs.dtu.dk/services/NetOGlyc/ (Hansen et al., 1998Go). Despite the high level of accuracy reported, predictions must be tested in relevant cell backgrounds to verify whether O-glycosylation occurs. For example, Nehrke et al. (1997)Go demonstrated that several reporter substrates predicted not to be glycosylated by NetOGlyc do acquire O-glycans in specific cell backgrounds. Gerken et al. (1997)Go demonstrated that none of three available predictive approaches were capable of successfully predicting the pattern of O-glycosylation experimentally determined in porcine submandibular gland mucin. Similarly, Thr126 and Ser152 of human insulin-like growth factor binding protein 6 are predicted not to acquire O-linked GalNAc by NetOGlyc, but these sites were determined experimentally to be occupied in the native protein (Neumann et al., 1998Go).

In vitro assays with synthetic peptide substrates and individual ppGaNTase isoforms. Because the ratio of the different transferases varies with cell and tissue type, it is not possible to dissect isoform-specific contributions using tissue or cell homogenates. Furthermore, other enzymatic activities in a homogenate may mask or mimic ppGaNTase activity, depending on the assay employed to detect GalNAc incorporation (Soudan et al., 1998Go). Thus a number of studies have been conducted with purified native or recombinant ppGaNTases to obtain insight into the substrate specificities of the different isoforms of this enzyme family. Libraries of structurally related peptides, derived from mucin glycoproteins (Wang et al., 1992Go, 1993Go), human von Willebrand factor (O'Connell et al., 1992Go) and erythropoietin (O'Connell et al., 1992Go; Wang et al., 1993Go; Yoshida et al., 1997Go; Elhammer et al., 1999Go) have been used to challenge the ppGaNTase isoforms. Results obtained corroborate earlier findings with tissue extracts—flanking sequence does influence acquisition of O-glycans, but no consensus rules emerge. In particular, charged residues at positions -1 and +3 (relative to the glycosylation sites) appear inhibitory to single-site glycosylation, whereas the presence of proline at position +3 appears to enhance the likelihood of glycosylation of the site. Most recently it has been argued that ppGaNTases glycosylate both threonine and serine residues, although threonine is modified with much higher catalytic efficiency (Elhammer et al., 1999Go). Analysis of native glycoproteins certainly demonstrates that O-linked serine residues exist, even though synthetic peptides containing the serinyl residue may fail to be glycosylated (for example, see Müller et al., 1997Go, who localized O-glycosylation sites from lactation associated MUC1).

Variation in assay conditions and the peptide substrate sequences (e.g., compare the sequences of the widely used peptide substrate EA2 that was derived from the tandem repeat region of the rat submandibular gland in the Tabak [PTTDSTTPAPTTK] and Clausen [DSTTPAPTTK] groups respectively) make it difficult to directly compare data across laboratories. Nevertheless, comparison of apparent Km values gives some insight into the synthetic substrate preferences of different isoforms (Table IV).


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Table IV. Apparent Km values (mM) for selected peptide substrates

 
A number of isoform-selective substrates have been identified by the Clausen group (Table V), but it is possible that as yet uncharacterized isoforms will be able to use these substrates as well. To date, only one isoform-selective substrate has been studied in a cell background. Nehrke et al. (1998)Go demonstrated that COS7 cells lack appreciable levels of ppGaNTase-T3 and are thus unable to glycosylate the substrate RGPGRAFVTIGKIGNMR efficiently (Bennett et al., 1996Go). Transfection of recombinant ppGaNTase-T3 into COS7 cells greatly enhanced the O-glycosylation of this peptide and the related RGPGRAFVSIGKIGNMR substrate, but transfection of ppGaNTase-T1 did not.


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Table V. Isoform-selective substrates for recombinant ppGaNTases

 
The sequence of GalNAc addition into multisite peptide substrates has been studied using recombinant ppGaNTase-T1, -2, -3, -4, -7, and -9 (now designated -T10) (Bennett et al., 1998Go; Ten Hagen et al., 1999Go, 2001Go; Iida et al, 1999Go; Tetaert et al, 2001aGo,bGo; Hanisch et al., 2001bGo; Kato et al., 2001aGo,bGo). Isoform-mediated site specificity is observed, although in some instances the sequence of incorporation appears random, whereas in other situations it is ordered.

Studies conducted to date have used a small number of substrates, and those employed tend to be relatively short peptides. Several emerging technologies may make it feasible to greatly expand the analysis of the "substrate space." Peptide arrays have found considerable use for epitope and paratope mapping and have recently been employed to assay kinase activity (reviewed Reineke et al., 2001Go). The major limitation of this approach is the relatively low affinities of ppGaNTases for substrates (typically in the high µM/low mM range). Fang et al. (2002)Go recently reported a method to create protein arrays in the presence of lipids. Whether the presence of lipids will modulate ppGaNTase activity is not yet known, but this issue can be addressed in future work using this method.

Analysis of higher order structure effects on O-glycosylation. A potential shortcoming of using synthetic peptides as substrates is that they lack discernible secondary and tertiary structures. Based on secondary structure predictions of tryptic fragments of ovine submandibular gland apomucin, Hill et al. (1977)Go suggested that the lack of secondary structure leads to increased accessibility to a partially purified ppGaNTase, most likely ppGaNTase-T1. Nishimori et al. (1994a)Go compared the glycosylation of one to five tandem repeats derived from MUC1 and found no discernible influence on O-glycan acquisition using tumor cell line extracts as the ppGaNTase source. They conclude that the higher-order secondary structure observed with multiple MUC1 tandem repeats (Fontenot et al., 1993Go) does not affect the acquisition of O-linked GalNAc. O'Connell et al. (1991)Go analyzed secondary structure of peptide substrates by circular dichroism spectroscopy and found that peptides with a random structure were glycosylated by partially purified ppGaNTase-T1, except when they contained a charged residue at position -1 relative to the glycosylation site. Kirnarsky et al. (1998)Go attempted to correlate the glycosylation of two peptide substrates (GVTSAPDTR, derived from the MUC1 tandem repeat sequence and a variant of this peptide -GVTSAGDTR) with the structural features of these peptides using recombinant ppGaNTase-T1, -T2, and -T3. They note that although ppGaNTase-T2 is able to glycosylate GVTSAPDTR when presented as part of a larger sequence context (Wandall et al., 1997Go) it fails to glycosylate the nonapeptide. ppGaNTase-T1 and -T3 each glycosylate GVTSAPDTR. By nuclear magmetic resonance, the peptide GVTSAPDTR assumes an extended ß-like conformation; substitution of G for P reduced the propensity to form an extended structure. This "mutated" peptide was a very poor substrate for all three transferases examined.

The influence of native conformation on the acquisition of O-glycans has been explored using erythropoietin as a model substrate (Elliot et al., 1994Go). They found that O-glycosylation can be altered by both mutations adjacent to the site of glycosylation as well as distant mutations that affect protein folding. Thus, primary, secondary, and tertiary protein structure appears to contribute to the efficiency of modification with O-glycans.

In vitro O-glycosylation of multisite substrates can proceed in a hierarchical manner. Prior glycosylation of specific serines and threonines in peptide substrates can influence sugar acquisition of neighboring positions (Brockhausen et al., 1990Go, 1996Go) in vitro. Thus, the density of glycosylation may be influenced in part by competing ppGaNTases and glycosyltransferases involved in formation of O-glycan core structures, such as ß-3-galactosyltransferase (core 1) and ß-6-glucosaminyltransferase (core 2). Hanisch et al. (1999)Go studied the influence of mono- and disaccharide substituted peptides derived from the MUC1 tandem repeat. They found positive regulatory effects of preexisting GalNAc on the subsequent addition of GalNAc at other sites. However, the presence of core 1 modified GalNAcs on the glycopeptide substrates had, in general, negative effects on the ability of a mixture of ppGaNTase isoforms to modify additional positions with GalNAc (Hanisch et al., 1999Go). Also, the presence of Galß1, 3GalNAc at varying positions of peptide (and glycopeptide) substrates derived from MUC1, MUC2, and MUC4 tandem repeat regions, resulted in partial or total inhibition of subsequent ppGaNTase-T2 or -T4 activity (Hanisch et al., 2001bGo).

Bennett et al. (1998)Go first reported that ppGaNTase-T4 was able to "complement" the activity of other ppGaNTases to complete the in vitro O-glycosylation of the glycopeptide T*APPAHGVT*S10APDT14RPAPGS*-T*APPA (where T* or S* contained or did not contain an {alpha}-GalNAc residue) by adding GalNAc to S10 and T 14 (Table VI). Thus, although ppGaNTase-T4 was first demonstrated to add GalNAc to peptide substrates (Hagen et al., 1997Go) and was subsequently shown to add GalNAc to a variety of other peptide substrates (Bennett et al., 1999aGo) (summarized in Table IV), this isoform could use a glycopeptide substrate as well.


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Table VI. Substrate preferences for selected peptide and glycopeptide substrates

 
More recently, we have determined that ppGaNTase-T7 and -T9 (now -T10) are each glycopeptide transferases (gpGaNTases) and appear to require the prior addition of GalNAc to a peptide before it can be used as a substrate (Ten Hagen et al., 1999Go, 2001Go; also see Bennett et al., 1999bGo). There appears to be a preferential addition of successive GalNAc residues N-terminal to those already present (Ten Hagen et al., 2001Go; Tetaert et al., 2001aGo,bGo). Thus, site- specific O-glycosylation by several ppGaNTases appears to be influenced by the position and structure of previously added O-glycans.

Presently there appear to be two types of glycopeptide ppGaNTases—those that act on both peptide substrates and glycopeptides (e.g., ppGaNTase-T4) and those that appear to act only on glycopeptide substrates (e.g., ppGaNTase-T9, now designated -T10). Nevertheless, this distinction may prove to be inaccurate because it is possible that a peptide substrate will be found that glycopeptide transferases, such as ppGaNTase-T10, will directly glycosylate.

Collectively, these observations argue in favor of a hierarchical addition of core GalNAc residues to apomucins, implying that the complete glycosylation of certain substrates is dependent on the coordinated action of multiple family members.

In a technical tour de force, Gerken and colleagues mapped the oligosaccharide distribution of O-glycans attached to the porcine submandibular gland mucin tandem repeat (Gerken et al., 1997Go, 1998Go, 2002Go). They find that the core 1 structure Galß1,3GalNAc{alpha} is more likely to occur in less dense regions of O-glycosylation. In contrast, the addition of {alpha}1,2 Fuc shows no correlation with hydroxyamino acid density although there is a marked enrichment of fucosylated structures attached to serine residues compared to threonine. From these data it appears that hydroxyamino acid spacing contributes to O-glycan extension. Variation in oligosaccharide structure, called microheterogeneity, has been attributed to biosynthetic leakiness for the most part. However, if O-glycan extension is influenced by hydroxamino acid density, then it is more likely that the structural variation is contrived rather than spurious.

ppGaNTase isoforms are localized differentially within cellular compartments. Moreira et al. (1989)Go demonstrated by quantitative analysis of labeling with a monoclonal antibody with specificity for {alpha}-GalNAc residues that GalNAc is not observed over the rough ER but rather in the Golgi apparatus of rat submandibular glands. This suggests that in the mucin-producing rat submandibular gland the ppGaNTases localize somewhere in the Golgi apparatus. ppGaNTase has been localized by immunoelectron microscopy using a polyclonal antibody raised against purified ppGaNTase from porcine submandibular glands (Roth et al., 1994Go). Specific labeling was observed over the cis-Golgi, suggesting that addition of GalNAc begins within the most proximal Golgi compartment. However, it is not known if the polyclonal antibody employed cross-reacts with multiple ppGaNTase isoforms or if the assay employed is sufficiently sensitive to detect lower levels of ppGaNTase. Röttger et al. (1998)Go have compared the intracellular localization of endogenous ppGaNTase-T2 and epitope-tagged ppGaNTases-T1, -T2, and -T3. Epitope-tagged ppGaNTase-T1 distributed evenly throughout the Golgi stack, whereas endogenous and epitope-tagged ppGaNTase-T2 and epitope-tagged ppGaNTase-T3 were each about twice as abundant in the trans and medial stacks, respectively, relative to the cis compartment. It is not clear if the overexpression of the epitope-tagged isoforms influenced the subcellular localization of the enzymes. Although these data suggest that initiation may not be confined to the cis-Golgi compartment, antibodies with the requisite specificity to discriminate among the native ppGaNTase isoforms will have to be employed to determine if different ppGaNTases localize in discrete subcellular compartments. Regardless, as pointed out in a recent review (Opat et al., 2001Go), Golgi proteins appear to recycle among the cisternae and between the ER and the Golgi apparatus. Taken together with the hierarchical glycosylation pattern observed in vitro with peptide and glycopeptide substrates, it is possible that enzyme localization within the Golgi may provide additional regulatory constraints governing which positions within a substrate are glycosylated and in what order. It remains to be determined how enzyme localization is regulated.

Genetic systems to analyze ppGaNTase function

Progress has been made toward ablating the expression of specific ppGaNTases in mouse models. Hennet et al. (1995)Go ablated expression of a highly homologous form of ppGaNTase-T1 (which is now designated ppGaNTase-T13; Zhang et al., 2002Go). Homozygous nulls are fertile and develop normally yet show reduced Tn antigen expression in the cerebellum. The transcript for this isoform is most highly expressed within the brain. Cre-lox recombination has been used to generate mice lacking exon 3 of ppGaNTase-T1 (Westerman et al., 1999Go). Homozygous nulls are fertile and appear to develop normally. However, using a -T1 selective substrate, they lack significant ppGaNTase-T1 activity in the spleen, kidney, thymus, liver, submandibular gland, and lung. Variation in the O-glycan lectin binding profile of lymphocyte subsets was observed, and the consequences of this are currently under investigation. We have recently succeeded in generating chimeric mice containing ablations of ppGaNTase-T4 and -T5. Homozygous nulls of -T4 and -T5 are fertile and appear to develop normally.

Although there is great power in transgenic mouse models to study protein function, important caveats have been raised. Brookfield (1992)Go, Erickson (1993)Go, and Pearson (2002)Go have outlined reasons why many gene knockouts appear to have no phenotype. First, the gene ablated may not have a vital function. Second, there may be a compensatory up- (or down-) regulation of other genes particularly in instances of multigene families. Indeed, Routtenberg (1995)Go has argued that "knockout" animals are not "animals without that protein" but organisms that "respond to the mutation." Variable results may also be seen among different mouse strains (Pearson, 2002Go). Third, there may be coexpression of proteins with duplicated function. This is certainly plausible given the size of the ppGaNTase family and the substrate overlaps observed among the family members. Thus, it may be necessary to knockout two (or more?) ppGaNTases in mice before a phenotype can be observed. For example, in S. cerevisiae, the transfer of mannose residues in O-glycosidic linkage to serine or threonine is catalyzed by one of seven mannosyltransferases (PMT1–7). Gene disruption of two or more pmt genes encoding the O-mannosyltransferases is required before a resultant growth defect is observed (Gentzsch and Tanner, 1996Go). However, recent studies in D. melanogaster have demonstrated that at least one ppGaNTase, pgant35A, is required for viability, as mutations within the coding region of this gene (l(2)35A or pgant35A) result in a recessive lethal phenotype (Ten Hagen and Tran, 2002Go; Schwientek et al., 2002Go). Therefore, it remains possible that certain mammalian isoforms may be absolutely required as well, and we are currently working toward ablating the murine orthologue (termed ppGaNTase-T11) of Drosophila pgant35A.

O-Glycan pathobiology revisited

Several examples of O-glycan pathobiology have been reported that require reexamination in view of the finding that there are multiple ppGaNTases. One such example is the underglycosylation of the IgA1 hinge region that is hypothesized to play a key role in IgA nephropathy (Hiki et al., 1999Go, 2001Go; Tomana et al., 1999Go; Iwase et al., 2001Go). There is no evidence of any nucleotide sequence alteration or transcriptional change in the {alpha}1 hinge region (Greer et al., 1998Go). Rather, the alteration is thought to be posttranslational in nature. Mattu et al. (1998)Go determined that O-glycans are normally located at T228, S230, and S232, whereas sites T225 and T236 are partially occupied in the sequence PST225PPTPSPSTPPTPSPS. Hiki et al. (2001)Go determined that the hinge region of IgA1 is underglycosylated in IgA nephropathy patients compared to healthy subjects. We propose that the underglycosylation observed in IgA mediated nephropathy may be the result of lowered expression or absence of a particular ppGaNTase isoform necessary to complete the O-glycosylation. Indeed, ppGaNTase-T2 has recently been shown to transfer GalNAc to the appropriate residues in vitro (Iwasaki et al., 2002Go).

The clustering of O-glycans appears to have functional significance. The presence of an O-glycan "collar" is thought to extend the length of a polypeptide backbone and is often observed in cell surface receptors (Jentoft, 1990Go). O-glycans also serve as the recognition site of many ligands including selectins (Yeh 2001Go). Hakomori (2002)Go recently proposed that membrane receptors decorated with O-glycans form an association with signal transducers in cholesterol-rich microdomains (the "glycosynapse"). Densely glycosylated domains are less susceptible to proteolytic digestion; for this reason mucins are able to form a protective blanket on mucosal surfaces (Tabak, 1995Go). Epitope clustering has been well documented; for example, Ley structures clustered on three successive serine residues were more effective at eliciting reactive antibodies than a construct substituted with a single Ley oligosaccharide (Kudryashov et al., 2001Go). Horan et al. (1999)Go presented intriguing findings that demonstrate a change in the selectivity of a lectin for carbohydrates when presented at different densities. They speculate that the same protein could trigger two different biological responses by binding differentially to two different carbohydrate ligands. Because different ppGaNTases are required to fully glycosylate different subsets of hydroxyamino acids, it follows that altered levels of specific ppGaNTases could lead to the expression of a glycosylated species with altered function.

The low-density lipoprotein (LDL) receptor consists of five discrete domains: the N-terminal domain, the site of ligand binding, is cysteine-rich; the second domain is EFG precursor-like; the third domain contains a mucin-like region; the fourth domain is the transmembrane spanning region; and the final domain is a short cytoplasmic tail. The receptor is not functionally expressed in the UDP-Gal/UDP-GalNAc 4-epimerase deficient Chinese hamster ovary (CHO) cell line (Kingsley et al., 1986Go), unless the cells are cultivated under conditions that allow O-glycosylation to proceed uninhibited. Subsequently, Davis et al. (1986)Go demonstrated domain 3 was not required for the LDL receptor to be functionally expressed in wild-type CHO cells suggesting that O-glycans found in the other domains may play the critical role in stabilizing receptor expression/function. When expressed in the monensin- resistant CHO cell line 31 (Monr31), the LDL receptor has lowered binding affinity (Segushi et al., 1991Go). Analysis revealed that O-linked oligosaccharides found outside of the clustered region (domain three) were lacking in LDL receptors expressed in the Monr31 cells. Considered together, this suggests that specific O-glycans not associated with domain 3 of the LDL receptor are necessary for normal receptor function/binding. The absence of a unique ppGaNTase in the Monr31 cell line was offered as one possible mechanism underlying the functional defects observed (Segushi et al., 1991Go). This could be re-examined now in light of our current knowledge of the ppGaNTase family.

Conclusions

As the human and mouse genome projects reach their conclusion, we will have a clearer understanding of the full complement of mammalian ppGaNTases. Given the ease with which genetic screens can be accomplished in model organisms such as C. elegans and D. melanogaster, it is likely that future advances in establishing functional roles for different ppGaNTases will first emerge using these and other model organisms. The next challenge will be to define the hierarchical network of glycosyltransferase interactions in vivo and to identify the repertoire of substrates that are glycosylated by each isoform. Only then can a complete understanding of the biological function played by this glycosyltransferase family be reached.

Acknowledgements

We thank Dr. William Young for his careful reading of the manuscript. L.A.T. thanks current and past members of our laboratory group for their many contributions.


    Footnotes

1 To whom correspondence should be addressed; e-mail: Lawrence.Tabak{at}nih.gov Back

Abbreviations

CHO, Chinese hamster ovary; ER, endoplasmic reticulum; GT1, glycosyltransferase motif 1; LDL, low-density lipoprotein; MYA, million years ago; ppGaNTase, UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase.

References

Alfalah, M., Jacob, R., Preuss, U., Zimmer, K.-P., Naim, H., and Naim, H.Y. (1999) O-linked glycans mediate apical sorting of human intestinal sucrase-isomaltase through association with lipid rafts. Curr. Biol., 9, 593–596.[CrossRef][ISI][Medline]

Apweiler, R., Hermjakob, H., and Sharon, N. (1999) On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database. Biochim. Biophys. Acta, 1473, 4–8.[ISI][Medline]

Altschuler, Y., Kinlough, C.L., Poland, P.A., Bruns, J.B., Apodaca, G., Weisz, O.A., and Hughey, R.P. (2000) Clathrin-mediated endocytosis of MUC1 is modulated by its glycosylation state. Mol. Biol. Cell, 11, 819–831.[Abstract/Free Full Text]

Bennett, E.P., Hassan, H., and Clausen, H. (1996) cDNA cloning and expression of a novel human UDP-N-acetyl-{alpha}-D-galactosaminyltransferase. J. Biol. Chem., 271, 17006–17012.[Abstract/Free Full Text]

Bennett, E.P., Hassan, H., Mandel, U., Mirgorodskaya, E., Roepstorff, P., Burchell, J., Taylor-Papadimitriou, J., Hollingsworth, M.A., Merkx, G., van Kessel, A.G., and others. (1998) Cloning of a human UDP-N-Acetyl-{alpha}-D-Galactosamine:polypeptide N-acetylgalactosaminyltransferase that complements other Ga1Nac-transferases in complete O-glycosylation of the MUC1 tandem repeat. J. Biol. Chem., 273, 30472–30481.[Abstract/Free Full Text]

Bennett, E.P., Hassan, H., Mandel, U., Hollingsworth, M.A., Akisawa, N., Ikematsu, Y., Merkx, G., van Kessel, A.G., Olofsson, S., and Clausen, H. (1999a) Cloning and characterization of a close homologue of human UDP-N-acetyl-{alpha}-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-T3, designated Ga1Nac-T6. J. Biol. Chem., 274, 25362–25370.[Abstract/Free Full Text]

Bennett, E.P., Hassan, H., Hollingsworth, M.A., and Clausen, H. (1999b) A novel human UDP-N-acetyl-D-Galactosamine:polypeptide N-Acetylgalactosaminyltransferase, GalNac-T7, with specificity for partial GalNAc-glycosylated acceptor substrates. FEBS Lett., 460, 226–230.[CrossRef][ISI][Medline]

Boix, E., Swaminathan, G.J., Zhang, Y., Natesh, R., Brew, K., and Acharya, K.R. (2001) Structure of UDP complex of UDP-galactose: {alpha}-Galactoside-{alpha}-1, 3-galactosyltransferase at 1.53-Å resolution reveals a conformational change in the catalytially important C terminus. J. Biol. Chem., 276, 48608–48614.[Abstract/Free Full Text]

Bourne, Y. and Henrissat, B. (2001) Glycoside hydrolases and glycosyltransferases: families and functional modules. Curr. Opin. Struct. Biol., 11, 593–600.[CrossRef][ISI][Medline]

Breton, C., Mucha, J., and Jeanneau, C. (2001) Structural and functional features of glycosyltransferases. Biochimie, 83, 713–718.[CrossRef][ISI][Medline]

Breuza, L., Garcia, M., Delgrossi, M.-H., and Le Bivic, A. (2002) Role of the membrane-proximal O-glycosylation site in sorting of the human receptor forneurotrophins to the apical membrane of MDCK cells. Exp. Cell Res., 273, 178–186.[CrossRef][ISI][Medline]

Briand, J.P., Andrews, S.P. Jr., Cahill, E., Conway, N.A., and Young, J.D. (1981) Investigation of the requirements for O-glycosylation by bovine submaxillary gland UDP-N-acetylgalactosamine:polypeptide N- acetylgalactosamine transferase using synthetic peptide substrates. J. Biol. Chem., 256, 12205–12207.[Abstract/Free Full Text]

Brockhausen, I., Möller, G., Merz, G., Adermann, K., and Paulsen, H. (1990) Control of mucin synthesis: the peptide portion of synthetic O-glycopeptide substrates influences the activity of O-glycan core 1 UDPgalactose:N-Acetyl-{alpha}-galactosaminyl-R ß3-galactosyltransferase. Biochem. J., 29, 10206–10212.

Brockhausen, I., Toki., D., Brockhausen, J., Peters, S., Bielfeldt, T., Kleen, A., Paulsen, H., Meldal, M., Hagen, F., and Tabak, L.A. (1996) Specificity of O-glycosylation by bovine colostrum UDP-GalNAc: polypeptide {alpha}-N-acetylgalactosaminyltransferase using synthetic glycopeptide substances. Glycoconj. J., 13, 849–856.[ISI][Medline]

Brookfield, J. (1992) Can genes be truly redundant? Curr. Biol., 2, 553–554.

Cai, Y.-D., Yu, H., and Chou, K.-C. (1997) Artificial neural network method for predicting the specificity of GalNAc-transferase. J. Prot. Chem., 16, 689–700.[ISI][Medline]

Cai, Y.-D., Liu, X.-J., Xu, X.-B., and Chou K.-C. (2002) Support vector machines for predicting the specificity of GalNAc-transferase. Peptides, 23, 205–208[CrossRef][ISI][Medline]

Campbell, J.A., Davies, G.J., Bulone, V., and Henrissat, B. (1997) A classification of nucleotide-diphospho-sugar glycosyltransferases based on amino acid sequence similarities. Biochem. J., 326, 929–942.[ISI][Medline]

Campbell, J.A., Davies, G.J., Bulone, V., and Henrissat, B. (1998) Correction: a classification of nucleotide-diphospho-sugar glycosyltransferases based on amino acid sequence similarities. Biochem. J., 329, 719.[ISI][Medline]

Christlet, T.H.T. and Veluraja, K. (2001) Database analysis of O-glycosylation sites in proteins. Biophys. J., 80, 952–960.[Abstract/Free Full Text]

Cottrell, J.M., Hall, R.L., Sturton, R.G., and Kent, P.W. (1992) Polypeptide N-acetylgalactosaminyltransferase activity in tracheal epithelial microsomes. Biochem. J., 283, 299–305.[ISI][Medline]

Coutinho, P.M. and Henrissant, B. (1999) Carbohydrate-active enzymes server. Available online at http://afmb.cnrs-mrs.fr/~cazy/CAZY/ index.html.

Czeszak, X., Ricart, G., Tetaert, D., Michalski, J.C., and Lemoine, J. (2002) Identification of substituted sites MUC5AC mucin motif peptides after enzymatic O-glycosylation combining ß-elimination and fixed-charge derivatization. Rapid Commun. Mass Spectrom., 16, 27–34.[CrossRef][ISI][Medline]

Davies, G.J. (2001) Sweet secrets of synthesis. Nat. Struct. Biol., 8, 98–100.[CrossRef][ISI][Medline]

Davis, C.G., Elhammer, A., Russell, D.W., Schneider, W.J., Kornfeld, S., Brown, M.S., and Goldstein, J.L. (1986) Deletion of clustered O-linked carbohydrates does not impair function of low density lipoprotein receptor in transfected fibroblasts. J. Biol. Chem., 261,2828–2838.[Abstract/Free Full Text]

De Haan, C.A.M., Roestenberg, P., de Wit, M., and de Vries, A.A.F. (1998) Structural requirements for O-glycosylation of the mouse hepatitis virus membrane protein. J. Biol. Chem., 273, 29905–29914.[Abstract/Free Full Text]

Dürr, G., Strayle, J., Plemper, R., Elbs, S., Klee, S.K., Catty, P., Wolf, D.H., and Rudolph, H.K. (1998) The medial-Golgi ion pump Pmr1 supplies the yeast secretory pathway with Ca2+ and Mn2+ required for glycosylation, sorting, and endoplasmic reticulum-associated protein degradation. Mol. Biol. Cell, 9, 1149–1162.[Abstract/Free Full Text]

Elhammer, Å.P. and Kornfeld, S. (1986) Purification and characterization of UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase from bovine colostrum and murine lymphoma BW5147 cells. J. Biol. Chem., 261, 5249–5255.[Abstract/Free Full Text]

Elhammer, Å.P., Poorman, R.A., Brown, E., Maggiora, L.L., Hoogerheide, J.G., and Kézdy, F.J. (1993) The specificity of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase as inferred from a database of in vivo substrates and from the in vitro glycosylation of proteins and peptides. J. Biol. Chem., 268, 10029–10038.[Abstract/Free Full Text]

Elhammer, Å.P., Kézdy, F.J., and Kurosaka, A. (1999) The acceptor specificity of UDP-GalNAc:polypeptide N-Acetylgalactosaminyltransferases. Glycoconj. J., 16, 171–180.[ISI][Medline]

Elliott, S., Bartley, T., Delorme, E., Derby, P., Hunt, R., Lorenzini, T., Parker, V., Rohde, M.F., and Stoney, K. (1994) Structural requirements for addition of O-linked carbohydrate to recombinant erythropoietin. Biochemistry, 33, 11237–11245.[ISI][Medline]

Erickson, H.P. (1993) Gene knockouts of c-src, transforming growth factor ß1, and tenascin suggest superfluous, nonfunctional expression of proteins. J. Cell Biol., 120, 1079–1081.[ISI][Medline]

Fang, Y., Frutos, A.G., and Lahiri, J. (2002) Membrane protein microarrays. J. Am. Chem. Soc., 124, 2394–2395.[CrossRef][ISI][Medline]

Fontenot, J.D., Tjandra, N., Bu, D., Ho, C., Montelaro, R.C., and Finn, O.J. (1993) Biophysical characterization of one-, two-, and three-tandem repeats of human mucin (muc-1) protein core. Cancer Res., 53, 5386–5394.[Abstract]

Furukawa, K., Takamiya, K., Okada, M., Inoue, J., Fukumoto, S., and Furukawa, K. (2001) Novel functions of complex carbohydrates elucidated by the mutant mice of glycosyltransferase genes. Biochim. Biophys. Acta, 1525, 1–12.[ISI][Medline]

Garner, B., Merry, A.H., Royle, L., Harvey, D.J., Rudd, P.M., and Thillet, J. (2001) Structure elucidation on the N- and O-glycans of human apolipoprotein(a). J. Biol. Chem., 276, 22200–22208.[Abstract/Free Full Text]

Gastinel, L.N., Cambillau, C., and Bourne, Y. (1999) Crystal structures of the bovine ß4galactosyltransferase catalytic domain and its complex with uridine diphosphogalactose. EMBO J., 18, 3546–3557.[Abstract/Free Full Text]

Gastinel, L.N., Bignon, C., Misra, A.K., Hindsgual, O., Shaper, J.H., and Joziasse, D.H. (2001) Bovine ß1, 3-galactosyltransferase catalytic domain structure and its relationship with ABO histo-blood group and glycosphingolipid glycosyltransferases. EMBO J., 20, 638–649.[Abstract/Free Full Text]

Gentzsch, M. and Tanner, W. (1996) The PMT family: protein O-glycosylation in Saccharomyces cerevisiae is vital. EMBO J., 15, 5752–5759.[Abstract]

Gerken, T.A., Owens, C.L., and Pasumarthy, M. (1997) Determination of the site-specific O-glycosylation pattern of the porcine submaxillary mucin tandem repeat glycopeptide. J. Biol. Chem., 272, 9709–9719.[Abstract/Free Full Text]

Gerken, T.A., Owens, C.L., and Pasumarthy, M. (1998) Site-specific core 1 O-glycosylation pattern of the porcine submaxillary gland mucin tandem repeat. J. Biol. Chem., 273, 26580–26588.[Abstract/Free Full Text]

Gerken, T.A., Gilmore, M., and Zhang, J. (2002) Determination of the site-specific oligosaccharide distribution of the O-glycans attached to the porcine submaxillary mucin tandem repeat. J. Biol. Chem., 277, 7736–7751.[Abstract/Free Full Text]

Gooley, A.A. and Williams, K.L. (1994) Towards characterizing O-glycans: the relative merits of in vivo and in vitro approaches in seeking peptide motifs specifying O-glycosylation sites. Glycobiology, 4, 413–417.[ISI][Medline]

Greer, M.R., Barratt, J., Harper, S.J., Allen, A.C., and Feehally, J. (1998) The nucleotide sequence of the IgA1 hinge region in IgA nephropathy. Nephrol. Dial. Transplant., 13, 1980–1983.[Abstract]

Guo, J.-M., Zhang, Y., Cheng, L., Iwasaki, H., Wang, H., Kubota, T., Tachibana, K., and Narimatsu, H. (2002) Molecular cloning and characterization of a novel member of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family, pp-GalNAc-T121. FEBS Lett., 26297, 1–8.

Hagen, F.K. and Nehrke, K. (1998) cDNA cloning and expression of a family of UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase sequence homologs from Caenorhabditis elegans. J. Biol. Chem., 273, 8268–8277.[Abstract/Free Full Text]

Hagen, F.K., VanWuyckhuyse, B., and Tabak, L.A. (1993) Purification, cloning, and expression of a bovine UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase. J. Biol. Chem., 268, 18960–18965.[Abstract/Free Full Text]

Hagen, F.K., Gregoire, C.A., and Tabak, L.A. (1995) Cloning and sequence homology of a rat UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase. Glycoconj. J., 12, 901–909.[ISI][Medline]

Hagen, F.K., Ten Hagen, K.G., Beres, T.M., Balys, M.M., VanWuyckhuyse, B.C., and Tabak, L.A. (1997) cDNA cloning and expression of a novel UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase. J. Biol. Chem., 272, 13843–13848.[Abstract/Free Full Text]

Hagen, F.K., Hazes B., Raffo, R., deSa, D., and Tabak, L. (1999) Structure–function analysis of the UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase. J. Biol. Chem., 274, 6797–6803.[Abstract/Free Full Text]

Hagen, F.K., Layden, M., Nehrke, K., Gentile, K., Berbach, K., Tsao, C.C., and Forsythe, M. (2001) Mucin-type O-glycosylation in C. elegans is initiated by a family of glycosyltransferases. Trends Glycosci. Glycotech., 13, 463–479.[ISI]

Hakomori, S. (2002) The glycosynapse. Proc. Natl Acad. Sci. USA, 99, 225–232.[Abstract/Free Full Text]

Hanisch, F.-G., Muller, S., Hassan, H., Clausen, H., Zachara, N., Gooley, A.A., Paulsen, H., Alving, K., and Peter-Katalinic, J. (1999) Dynamic epigenetic regulation of initial O-glycosylation by UDP-N-acetylgalactosamine:peptide N-acetylgalactosaminyltransferases. J. Biol. Chem., 274, 9946–9954.[Abstract/Free Full Text]

Hanisch, F.-G., Jovanovic, M., and Katalinic, J.P. (2001a) Glycoprotein identification and localization of O-glycosylation sites by mass spectrometric analysis of deglycosylated/alkylaminylated peptide fragments. Anal. Biochem., 290, 47–59.[CrossRef][ISI][Medline]

Hanisch, F.-G., Reis, C.A., Clausen, H., and Paulsen, H. (2001b) Evidence for glycosylation-dependent activities of polypeptide N- acetylgalactosaminyltransferases rGalNAc-T2 and -T4 on mucin glycopeptides. Glycobiology, 11, 731–740.[Abstract/Free Full Text]

Hansen, J.E., Lund, O., Tolstrup, N., Gooley, A.A., Williams, K.L., and Brunak, S. (1998) NetOglyc: prediction of mucin type O-glycosylation sites based on sequence context and surface accessibility. Glycoconj. J., 15, 115–130.[CrossRef][ISI][Medline]

Hassan, H., Reis, C.A., Bennett, E.P., Mirgorodskaya, E., Roepstorff, P., Hollingsworth, M.A., Burchell, J., Taylor-Papadimitriou, J., and Clausen, H. (2000) The lectin domain of UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-T4 directs its glycopeptide specificities. J. Biol. Chem., 275, 38197–38205.[Abstract/Free Full Text]

Hazes, B. (1996) The (QxW)3 domain: a flexible lectin scaffold. Protein Sci., 5, 1490–1501.[Abstract/Free Full Text]

Henle, K.J., Nagle, W.A., Bedford, J.S., and Harvey, W.F. (1990) Protein glycosylation in heat-sensitive and thermotolerance-deficient mutants of Chinese hamster ovary cells. J. Cell Sci., 95, 555–561.[Abstract]

Hennebicq, S., Tetaert, D., Soudan, B., Boersma, A., Briand, G., Richet, C., Gagnon, J., and Degand, P. (1998a) Influence of the amino acid sequence on the MUC5AC motif peptide O-glycosylation by human gastric UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase(s). Glycoconj. J., 15, 275–282.[CrossRef][ISI][Medline]

Hennebicq, S., Tetaert, D., Soudan, B., Briand, G., Richet, C., Demeyer, D., Gagnon, J., Petillot, Y., and Degand, P. (1998b) Polypeptide N-acetylgalactosaminyltransferase activities towards the mucin MUC5AC peptide motif using microsomal preparations of normal and tumoral digestive mucosa. Biochimie, 80, 69–73.[CrossRef][ISI][Medline]

Hennet, T., Hagen, F.K., Tabak, L.A., and Marth, J.D. (1995) T-cell-specific deletion of a polypeptide N-acetylgalactosaminyltransferases gene by site-directed recombination. Proc. Natl Acad. Sci. USA, 92, 12070–12074.[Abstract]

Hiki, Y., Kokubo, T., Iwase, H., Masaki, Y., Sano, T., Tanaka, A., Toma, K., Hotta, K., and Kobayashi Y. (1999) Underglycosylation of IgA1 hinge plays a certain role for its glomerular deposition in IgA nephropathy. J. Am. Soc. Nephrol., 10, 760–769.[Abstract/Free Full Text]

Hiki, Y., Odani, H., Takahashi, M., Yasuda, Y., Nishimoto, A., Iwase, H., Shinzato, T., Kobayashi, Y., and Maeda, K. (2001) Mass spectrometry proves under-O-glyccosylation of glomerula IgA1 in IgA nephropathy. Kidney Intl., 59, 1077–1085.[CrossRef][ISI][Medline]

Hill, H.D. Jr., Schwyzer, M., Steinman, H.M., and Hill, R.L. (1977) Ovine submaxillary mucin. J. Biol. Chem., 252, 3799–3804.[Abstract]

Hogenesch, J.B., Ching, K.A., Batalov, S., Su, A.I., Walker, J.R., Zhou, Y., Kay, S.A., Schultz, P.G., and Cooke, M.P. (2001) A comparison of the celera and ensembl predicted gene sets reveals little overlap in novel genes. Cell, 106, 413–415.[ISI][Medline]

Homa, F.L., Hollander, T., Lehman, D.J., Thomsen, D.R., and Elhammer, Å.P. (1993) Isolation and expression of a cDNA clone encoding a bovine UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase. J. Biol. Chem., 268, 12609–12616.[Abstract/Free Full Text]

Hooper, L.V. and Gordon, J.I. (2001) Glycans as legislators of host-microbial interactions: spanning the spectrum from symbiosis to pathogenicity. Glycobiology, 11, 1R–10R.[Abstract/Free Full Text]

Hooper, N.M., Karran, E.H., and Turner, A.J. (1997) Membrane protein secretases. Biochem. J., 321, 265–279.[ISI][Medline]

Horan, N., Yan, L., Isobe, H., Whitesides, G.M., and Kahne, D. (1999) Nonstatistical binding of a protein to clustered carbohydrates. Proc. Natl Acad. Sci. USA, 96, 11782–11786.[Abstract/Free Full Text]

Hughes, R.C., Bradbury, A.F., and Smyth. D.G. (1988) Substrate recognition by UDP-N-acetyl-{alpha}-D-galactosamine: polypeptide N-acetyl-{alpha}-D-galactosaminyltransferase. Effects of chain length and disulphide bonding of synthetic peptide substrates. Carbohydr. Res., 178, 259–269.[CrossRef][ISI][Medline]

Iida, S.-i., Takeuchi, H., Hassan, H., Clausen, H., and Irimura, T. (1999) Incorporation of N-acetylgalactosamine into consecutive threonine residues in MUC2 tandem repeat by recombinant human N-acetyl-D-galactosamine transferase-T1, T2, and T3. FEBS Lett., 449, 230–234.[CrossRef][ISI][Medline]

Iida, S.-i., Takeuchi, H., Kato, K., Yamamoto, K., and Irimura, T. (2000) Order and maximum incorporation of N-acetyl-D-galactosamine into threonine residues of MUC2 core peptide with microsome fraction of human-colon-carcinoma LS174T cells. Biochem. J., 347, 535–542.[CrossRef][ISI][Medline]

Imberty, A., Piller, V., Piller, F., and Breton, C. (1997) Fold recognition and molecular modeling of a lectin-like domain in UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases. Protein Eng., 10, 1353–1356.[Abstract]

Inoue, M., Yamashina, I., and Nakada, H. (1998) Glycosylation of the tandem repeat unit of the MUC2 polypeptide leading to the synthesis of the Tn antigen. Biochem. Biophys. Res. Commun., 245, 23–27.[CrossRef][ISI][Medline]

Inoue, M., Takahashi, S., Yamashina, I., Kaibori, M., Okumura, T., Kamiyama, Y., Vichier-Guerre, S., Cantacuzene, D., and Nakada, H. (2001) High density O-glycosylation of the MUC2 tandem repeat unit by N-acetylgalactosaminyltransferase-3 in colonic adenocarcinoma extracts. Cancer Res., 61, 950–956.[Abstract/Free Full Text]

Iwasaki, H., Zhang, Y., Tachibana, K., Gotoh, M., Kikuchi, N., Kwon, Y.-D., Togayachi, A., Kudo, T., Kubota, T., and Narimatsu, H. (2002) Initiation of O-glycan synthesis in IgAl hinge region is determined by a single enzyme, UDP-N-Acetyl-a-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase 2; pp-GalNAc-T2. J. Biol. Chem., forthcoming.

Iwase, H., Tanaka, A., Hiki, Y., Kokubo, T., Sano, T., Ishii-Karakasa, I., Hisatani, K., Kobayashi, Y., and Hotta, K. (2001) Analysis of the microheterogeneity of the IgA1 hinge glycopeptide having multiple O-linked oligosaccharides by capillary electrophoresis. Anal. Biochem., 288, 22–27.[CrossRef][ISI][Medline]

Jentoft, N. (1990) Why are proteins O-glycosylated? Trends Biochem. Sci., 15, 291–294.[CrossRef][ISI][Medline]

Kato, K., Takeuchi, H., Kanoh, A., Mandel, U., Hassan, H., Clausen, H., and Irimura, T. (2001a) N-acetylgalactosamine incorporation into a peptide containing consecutive threonine residues by UDP-N-acetyl-D-galactosaminide:polypeptide N-acetylgalactosaminyl-transferases. Glycobiology, 11, 821–829.[Abstract/Free Full Text]

Kato, K., Takeuchi, H., Miyahara, N., Kanoh, A., Hassan, H., Clausen, H., and Irimura, T. (2001b) Distinct orders of GalNAc incorporation into a peptide with consecutive threonines. Biochem. Biophys. Res. Commun., 287, 110–115.[CrossRef][ISI][Medline]

Kawaguchi, N., Ohmori, T., Takeshita, Y., Kawanishi, G., Katayama, S., and Yamada H. (1986) Occurrence of Gal ß1,3 Ga1NAc-Ser/Thr in the linkage region of polygalactosamine containing fungal glycoprotein from Cordyceps Ophioglossoides. Biochem. Biophys. Res. Commun., 140, 350–356.[ISI][Medline]

Kingsley, D.M., Kozarsky, K F., Hobbie, L., and Krieger M. (1986) Reversible defects in O-linked glycosylation and LDL receptor expression in a UDP-Gal/UDP-GalNAc 4-epimerase deficient mutant. Cell, 44, 749–759.[ISI][Medline]

Kingsley, P.D., Ten Hagen, K.G., Maltby, K.M., Zara, J., and Tabak, L.A. (2000) Diverse spatial expression patterns of UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase family members mRNAs during mouse development. Glycobiology, 10, 1317–1323.[Abstract/Free Full Text]

Kirnarsky, L., Nomoto, M., Ikematsu, Y., Hassen, H., Bennett, E.P., Cerny, R.L., Clausen, H., Hollingsworth, M.A., and Sherman, S. (1998) Structural analysis of peptide substance for mucin-type O-glycosylation. Biochemistry, 37, 12811–12817.[CrossRef][ISI][Medline]

Kitazume, S., Tachida, Y., Ritsuko, O., Shirotani, K., Saido, T.C., and Hashimoto, Y. (2001) Alzheimer's ß-secretase, ß-site amyloid precursor protein-cleaving enzyme, is responsible for cleavage secretion of a Golgi-resident sialyltransference. Proc. Natl Acad. Sci. USA, 98, 13554–13559.[Abstract/Free Full Text]

Kornberg, A. (1991) For the love of enzymes. Harvard University Press, London.

Kudryashov, V., Glunz, P.W., Williams, L.J., Hintermann S., Danishefsky, S. J., and Lloyd, K.O. (2001) Toward optimized carbohydrate-based anticancer vaccines: Epitope clustering, carrier structure, adjuvant all influence antibody responses to Lewisy conjugates in mice. Proc. Natl Acad. Sci. USA, 98, 3264–3269.[Abstract/Free Full Text]

Mandel, U., Hassan, H., Therkildsen, M.H., Rygaard, J., Jakobsen, M.H., Juhl, B.R., Dabelsteen, E., and Clausen, H. (1999) Expression of polypeptide GalNAc-transferases in stratified epithelia and squamous cell carcinomas: immunohistological evaluation using monoclonal antibodies to three members of the GalNAc-transferase family. Glycobiology, 9, 43–52.[Abstract/Free Full Text]

Marth, J.D. (1996) Complexity in O-linked oligosaccharide biosynthesis engendered by multiple polypeptide N-acetylgalactosaminyltransferases. Glycobiology, 6, 701–705.[ISI][Medline]

Mattu, T.S., Pleass, R.J., Willis, A.C., Kilian, M., Wormaldt M.R., Lellouch, A.C., Rudd, P.M., Woof, J.M., and Dwek, R.A. (1998) The glycosylation and structure of human serum IgA1, Fab, and Fc regions and the role of N-glycosylation on Fc{alpha} receptor interaction. J. Biol. Chem., 273, 2260–2272.[Abstract/Free Full Text]

McGuire, E.J. and Roseman, S. (1967) Enzymatic synthesis of the protein-hexosamine linkage in sheep submaxillary mucin. J. Biol. Chem., 242, 3745–3755.[Abstract/Free Full Text]

Meurer, J.A., Drong, R.F., Homa, F.L., Slightom, J.L., and Elhammer, Å.P. (1996) Organization of a human UDP-GalNAc:polypeptide, N-acetylgalactosamine gene and a related processed pseudogene. Glycobiology, 6, 231–241.[Abstract]

Mika, K., Shoko, N., Hisashi, N., and Naruya, S. (2001) The evolutionary history of glycosyltransferase genes. Trends Glycosci. Glycotech., 13, 147–155.[ISI]

Mirgorodskaya, E., Hassan, H., Clausen, H., and Roepstorff, P. (2001) Mass spectrometric determination of O-glycosylation sites using ß-elimination and partial acid hydrolysis. Anal. Chem., 73, 1263–1269.[CrossRef][ISI][Medline]

Moody, A.M., Chui, D., Reche, P.A., Priatel, J.J., Marth, J.D., and Reinherz, E.L. (2001) Developmentally regulated glycosylation of the CD8{alpha}ß coreceptor stalk modulates ligand binding. Cell, 107, 501–512.[ISI][Medline]

Moreira, J.E., Tabak, L.A., Bedi, G.S., Culp, D.J., and Hand, A.R. (1989) Light and electron microscopic immunolocalization of rat submandibular gland mucin glycoprotein and glutamine/glutamic acid-rich proteins. J. Histochem. Cytochem., 37, 515–528.[Abstract]

Müller, S. and Hanisch, F.-G. (2002) Recombinant MUC1 probe authentically reflects cell-specific O-glycosylation profiles of endogenous breast cancer mucin. J. Biol. Chem., 277, 26103–26112.[Abstract/Free Full Text]

Müller, S., Goletz, S., Packer, N., Gooley, A., Lawson, A.M., and Hanisch. F.-G. (1997) Localization of O-glycosylation sites on glycopeptide fragments from lactation-associated MUC1. J. Biol. Chem., 272, 24780–24793.[Abstract/Free Full Text]

Muramatsu, T. (2000) Essential roles of carbohydrate signals in development, immune response and tissue functions, as revealed by gene targeting. J. Biochem., 127, 171–176.[Abstract]

Naim, H.Y., Joberty, G., Alfalah, M., and Jacob, R. (1999) Temporal association of the N- and O-linked glycosylation events and their implication in the polarized sorting of intestinal brush border sucrase-isomaltasae, aminopeptidase N, and dipeptidyl peptidase IV. J. Biol. Chem., 25, 17961–17967.[CrossRef]

Nehrke, K., Hagen, F.K., and Tabak, L.A. (1996) Charge distribution of flanking amino acids influences O-glycan acquisition in vivo. J. Biol. Chem., 271, 7061–7065.[Abstract/Free Full Text]

Nehrke, K., Ten Hagen, K.G., Hagen, F.K., and Tabak, L.A. (1997) Charge distribution of flanking amino acids inhibits O-glycosylation of several single-site acceptors in vivo. Glycobiology, 7, 1053–1060.[Abstract]

Nehrke, K., Hagen, F.K., and Tabak, L.A. (1998) Isoform-specific O-glycosylation by murine UDP-GaINAc:polypeptide N-acetylgalactosaminyltransferase-T3, in vivo. Glycobiology, 8, 367–371.[Abstract/Free Full Text]

Neumann, G.M., Marinaro, J.A., and Bach, L.A. (1998) Identification of O-glycosylation sites and partial characterization of carbohydrate structure and disulfide linkages of human insulin-like growth factor binding protein 6. Biochem. J., 37, 6572–6585.[CrossRef]

Nishimori, I., Perini, F., Mountjoy, K.P., Sanderson, S.D., Johnson, N., Cerny, R.L., Gross, M.L., Fontenot, D., and Hollingsworth, M.A. (1994a) N-acetylgalactosamine glycosylation of MUC1 tandem repeat peptides by pancreatic tumor cell extracts. Cancer Res., 54, 3738–3744.[Abstract]

Nishimori, I., Johnson, N.R., Sanderson, S.D., Perini, F., Mountjoy, K., Cerny, R.L., Gross, M.L., and Hollingsworth, M.A. (1994b) Influence of acceptor substrate primary amino acid sequence on the activity of human UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase. J. Biol. Chem., 269, 16123–16130.[Abstract/Free Full Text]

Nomoto, M., Izumi, H., Ise, T., Kato, K., Takano, H., Nagatani, G., Shibao, K., Ohta, R., Imamura, T., Kuwano, M., and others. (1999) Structural basis for the regulation of UDP-N-acetyl- (-D-galactosamine:polypeptide N-acetylgalactosaminyl transferase-3 gene expression in adenocarcinoma cells. Cancer Res., 59, 6214–6222.[Abstract/Free Full Text]

O'Connell, B., Tabak, L.A., and Ramasubbu, N. (1991) The influence of flanking sequences on O-glycosylation. Biochem. Biophys. Res. Commun., 180, 1024–1030.[ISI][Medline]

O'Connell, B.C., Hagen, F.K., and Tabak, L.A. (1992) The influence of flanking sequence on the O-glycosylation of threonine in vitro. J. Biol. Chem., 267, 25010–25018.[Abstract/Free Full Text]

Oelmann, S., Stanley, P., and Gerardy-Schahn, R. (2001) Point mutations identified in Lec8 chinese hamster ovary glycosylation mutants that inactivate both the UDP-galactose and CMP-sialic acid transporters. J. Biol. Chem., 276, 26291–26300.[Abstract/Free Full Text]

Opat, A.S., van Vliet, C., and Gleeson, P.A. (2001) Trafficking and localisation of resident Golgi glycosylation enzymes. Biochimie, 83, 763–773.[CrossRef][ISI][Medline]

Pearson, H. (2002) Surviving a knockout blow. Nature, 415, 8–9.[CrossRef][ISI][Medline]

Persson, K., Ly, H.D., Dieckelmann, M., Wakarchuk, W.W., Withers, S.G., and Strynadka, N.C.J. (2001) Crystal structure of the retaining galactosyltransferase LgtC from Neisseria meningitides in complex with donor and acceptor sugar analogs. Nat. Struct. Biol., 8, 166–175.[CrossRef][ISI][Medline]

Reineke, U., Volkmer-Engert, R., and Schneider-Mergener, J. (2001). Applications of peptide arrays prepared by the SPOT-technology. Curr. Opin. Biotechnol., 12, 59–64.[CrossRef][ISI][Medline]

Roseman, S. (1970) The synthesis of complex carbohydrates by multiglycosyltranferase systems and their potential function in intercellular adhesion. Chem. Phys. Lipids, 5, 270–297.[CrossRef][ISI][Medline]

Roth, J., Wang, Y., Eckhardt, A.E., and Hill, R.L. (1994) Subcellular localization of the UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltranference-mediated O-glycosylation reaction in the submaxillary gland. Proc. Natl Acad. Sci. USA, 91, 8935–8939.[Abstract]

Röttger, S., White, J., Wandall, H.H., Olivo, J-C., Stark, A., Bennett, E.P., Whitehouse, C., Berger, E.G., Clausen, H, and Nilsson, T. (1998) Localization of three human polypeptide GalNAc-transferases in HeLa cells suggests initiation of O-linked glycosylation throughout the Golgi apparatus. J. Cell Sci., 111, 45–60.[Abstract/Free Full Text]

Routtenberg, A. (1995) Knockout mouse fault lines. Nature, 374, 314–315.[ISI][Medline]

Sauer, J., Sigurskjold, B.W., Christensen, U., Frandsen, T.P., Mirgorodskaya, E., Harrison, M., Roepstorff, P., and Svensson, B. (2000) Glucoamylase: structure/function relationships, and protein engineering. Biochim. Biophys. Acta, 1543, 275–293.[ISI][Medline]

Schwientek, T., Bennett, E.P., Flores, C., Thacker, J., Hollmann, M., Reis, C.A., Behrens, J., Mandel, U., Keck, B., Schäfer, M.A., and others. (2002) Functional conservation of subfamilies of putative UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases in Drosophila, caenorhabditis elegans and mammals. J. Biol. Chem., 277, 22623–22638.[Abstract/Free Full Text]

Segushi, T., Merkle R.K., Ono, M., Kuwano M., and Cummings, R.D. (1991) The dysfunctional LDL receptor in a monensin-resistant mutant of Chinese hamster ovary cells lacks selected O-linked oligosaccharides. Arch. Biochem. Biophys., 284, 245–256.[ISI][Medline]

Shibao, K., Izumi, H., Nakayama, Y., Ohta, R., Nagata, N., Nomoto, M., Matsuo K-I., Yamada, Y., Kitazato, K., Itoh, H., and Kohno, K. (2002) Expressions of UDP-N-Acetyl-{alpha}-D-galactosaminepolypeptide GalNAc N-acetylgalactosaminyl transferase-3 in relation to differentiation and prognosis in patients with colorectal carcinoma. Cancer, 94, 1939–1946.[CrossRef][ISI][Medline]

Somers, W.S., Tang, J., Shaw, G.D., and Camphausen, R.T. (2000) Insights into the molecular basis of leukocyte tethering and rolling revealed by structures of P- and E-selectin bound to Slex and PSGL-1. Cell, 103, 467–479.[ISI][Medline]

Sørensen, T., White, T., Wandall, H.H., Kristensen, A.K., Roepstorff, P., and Clausen, H. (1995) UDP-N-acetyl-{alpha}-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase. J. Biol. Chem., 270, 24166–24173.[Abstract/Free Full Text]

Soudan, B., Tetaert, D., Hennebico, S., Briand, G., Zerimech, F., Richet, C., Demeyer, D., Gagnon, J., Petillot, Y., and Degand, P. (1998) Dipeptidyl aminotransferase activity and in vitro O-glycosylation of MUC5AC mucin motif peptides by human gastric microsomal preparations. J. Peptide Res., 51, 346–354.[ISI][Medline]

Stadie, T.R.E., Chai, W., Lawson, A.M., Byfield, P.G.H., and Hanisch, F.-G. (1995) Studies on the order and site specificity of GalNAc transfer to MUC1 tandem repeats by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase from milk or mammary carcinoma cells. Eur. J. Biochem., 229, 140–147.[Abstract]

Strous, G.J.A.M. (1979) Initial glycosylation of proteins with acetylgalactosaminylserine linkages. Proc. Natl Acad. Sci. USA, 76, 2694–2698.[Abstract]

Sugiura, M., Kawasaki, T., and Yamashina, I. (1982) Purification and characterization of UDP-GalNAc:polypeptide N-acetylgalactosamine transferase from an ascites hepatoma, AH 66. J. Biol. Chem., 257, 9501–9507.[Abstract/Free Full Text]

Sutherlin, M.E., Nishimori, I., Caffrey, T., Bennett, E.P., Hassan, H., Mandel, U., Mack, D., Iwamura, T., Clausen. H., and Hollingsworth, M.A. (1997) Expression of three UDP-N-acetyl- {alpha}-D-galactosamine:polypeptide Ga1NAc N-acetylgalactosaminytransferases in adenocarcinoma cell lines. Cancer Res., 57, 4744–4748.[Abstract]

Tabak, L.A. (1995) In defense of the oral cavity: structure, biosynthesis, and function of salivary mucins. Annu. Rev. Physiol., 57, 547–64.[CrossRef][ISI][Medline]

Ten Hagen, K.G. and Tran, D.T. (2002) A UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase is essential for viability in Drosophila melanogaster. J. Biol. Chem., 277, 22616–22622.[Abstract/Free Full Text]

Ten Hagen, K.G., Hagen, F.K., Balys, M.M., Beres, T.M., Van Wuyckhuyse, B., and Tabak, L.A. (1998) Cloning and expression of a novel, tissue specifically expressed member of the UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase family. J. Biol. Chem., 273, 27749–27754.[Abstract/Free Full Text]

Ten Hagen, K.G., Tetaert, D., Hagen, F.K., Richet, C., Beres, T.M., Gagnon, J., Balys, M.M., Van Wuyckhuyse, B., Bedi, G.S., Degand, P., and Tabak, L.A. (1999) Characterization of a UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases that displays glycopeptide N-acetylgalactosaminyltransferases activity. J. Biol. Chem., 274, 27867–27874.[Abstract/Free Full Text]

Ten Hagen, K.G., Bedi, G.S., Tetaert, D., Kingsley, P.D., Hagen, F.K., Balys, M.M., Beres, T.M., Degand, P., and Tabak. L.A. (2001) Cloning and characterization of a ninth member of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases family, ppGaNTase-T9. J. Biol. Chem., 276, 17395–17404.[Abstract/Free Full Text]

Tenno, M., Toba, S., Kézdy, F.J., Elhammer, Å.P., and Kurosaka A. (2002) Identification of two cysteine residues involved in the binding of UDP-GalNAc to UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferasae 1 (GalNAc-T1). Eur. J. Biochem., 269, 4308–4316.[Abstract/Free Full Text]

Tetaert, D., Briand, G., Soudan, B., Richet, C., Demeyer, D., Boersma, A., and Degand, P. (1994) Analysis by electrospray mass spectrometry of glycopeptides from the in vitro O-glycosylation reaction using human mucin motif peptide. Anal. Biochem., 222, 409–416.[CrossRef][ISI][Medline]

Tetaert, D., Ten Hagen, K, G., Richet, C., Boersma, A., Gagnon, J., and Degand, P. (2001a) Glycopeptide N-acetylgalactosaminytransferase specificities for O-glycosylated sites on MUC5AC mucin motif peptides. Biochem. J., 357, 313–320.[CrossRef][ISI][Medline]

Tetaert, D., Richet, C., Gagnon, J., Boersma, A., and Degand, P. (2001b) Studies of acceptor site specificities for three members of UDP-GalNAc: N-acetykgalactosaminyltransferases by using a synthetic peptide mimicking the tandem repeat of MUC5AC. Carbohydr. Res., 333, 165–171.[CrossRef][ISI][Medline]

Toba, S., Tenno, M., Konishi, M., Mikami, T., Itoh, N., and Kurosaka, A. (2000) Brain-specific expression of a novel human UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (Ga1NAc-T9). Biochim. Biophys. Acta, 1493, 264–268.[ISI][Medline]

Tomana, M., Novak, J., Julian, B.A., Matousovic, K., Konecny, K., and Mestecky, J. (1999) Circulating immune complexes in IgA nephropathy consist of IgA1 with galactose-deficient hinge region and antiglycan antibodies. J. Clin. Invest., 104, 73–81.[Abstract/Free Full Text]

Unligil, U.M., Zhou, S., Yuwaraj, S., Sarkar, M., Schachter, H., and Rini, J. M. (2000) X-ray crystal structure of rabbit N-acetylglucosaminyltransferase I: catalytic mechanism and a new protein superfamily. EMBO J., 19, 5269–5280.[Abstract/Free Full Text]

Wandall, H.H., Hassan, H., Mirgorodsksya, E., Kristensen, A.K., Roepstorff, P., Bennett, E.P., Nielsen, P.A., Hollingsworth, M.A., Burchell, J., Taylor-Papadimitriou, J., and Clausen, H. (1997) Substrate specificities of three members of the human UDP-N-acetyl-{alpha}-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases family, GalNAc-T1, -T2, and -T3. J. Biol. Chem., 272, 23503–23514.[Abstract/Free Full Text]

Wang, Y., Abernethy, J.L., Eckhardt, A.E., and Hill, R.L. (1992) Purification and characterization of a GalNAc:polypeptide N-acetylgalactosaminyltransferases specific for glycosylation of threonine residues. J. Biol. Chem., 267, 12709–23716.[Abstract/Free Full Text]

Wang, Y., Agrwal, N., Eckhardt, A.E., Stevens, R.D., and Hill, R.L. (1993) The acceptor substrate of porcine submaxillary UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase is dependent on the amino acid sequences adjacent to serine and threonine residues. J. Biol. Chem., 268, 22979–22983.[Abstract/Free Full Text]

Westerman, E.L., Ellies, L.G., Hagen, F.K., Marek, K.W., Sutton-Smith, M., Dell, A., Tabak, L.A., and Marth, J.D. (1999) Selective loss of O-glycans in mice lacking polypeptide GalNAcT-1. Glycobiology, 9, 1121.

White, K.E., Lorenz, B., Evans, W.E., Meitinger, T., Strom, T.M., and Econs, M.J. (2000) Molecular cloning of a novel UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases, GalNAc-T8 and analysis as a candidate autosomal dominant hypophosphatemic rickets (ADHR) gene. Gene, 246, 347–356.[CrossRef][ISI][Medline]

White, T., Bennett, E.P., Takio, K., Sørensen, T., Bonding, N., and Clausen, H. (1995) Purification and cDNA cloning of a human UDP-N-acetyl-{alpha}-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase. J. Biol. Chem., 270, 24156–25165.[Abstract/Free Full Text]

Wiggins, C.A.R. and Munro, S. (1998) Activity of the yeast MNN1 (-1, 3-mannosyltransferase requires a motif conserved in many other families of glycosyltransferases. Proc. Natl Acad. Sci. USA, 95, 7945–7950.[Abstract/Free Full Text]

Wilson, I.B.H., Gavel, Y., and von Heijne, G. (1991) Amino acid distributions around O-linked glycosylation sites. Biochem. J., 275, 529–534.[ISI][Medline]

Winans, K.A. and Bertozzi, C.R. (2002) An inhibitor of the human UDP-GlcNAc 4-epimerase identified from a uridine-based library: a strategy to inhibit O-linked glycosylation. Chem. Biol., 9, 113–129.[CrossRef][ISI][Medline]

Withers, S.G., Anthony R., Warren, J., Street, I.P., Rupitz, K., Kempton, J.B., and Aebersold, R. (1990) Unequivocal demonstration of the involvement of a glutamate residue as a nucleophile in the mechanism of a "retaining" glycosidase. J. Am. Chem. Soc., 112, 5887–5889.[ISI]

Wragg, S., Hagen F.K., and Tabak, L.A. (1995) Kinetic analysis of a recombinant UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase. J. Biol. Chem., 270, 16947–16954.[Abstract/Free Full Text]

Wragg, S., Hagen, F.K., and Tabak, L.A. (1997) Identification of essential histidine residues in UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-T1. Biochem. J., 328, 193–197.[ISI][Medline]

Xu, Z. and Weiss, A. (2002) Negativ regulation of CD45 by differential homodimerization of the alternatively spliced isoforms. Nature Immunol., 3, 764–771.[CrossRef][ISI][Medline]

Yeh, J.-C., Hiraoka, N., Petryniak, B., Nakayama, J., Ellies, L.G., Rabuka, D., Hindsgaul, O., Marth, J.D., Lowe, J.B., and Fukuda, M. (2001) Novel sulfated lymphocyte homing receptors and their control by a core1 extension ß1, 3-N-acetylglucosaminyltransferase. Cell, 105, 957–969.[CrossRef][ISI][Medline]

Yoshida, A., Hara, T., Ikenaga, H., and Takeuchi, M. (1995) Cloning and expression of a porcine UDP-GalNAc:polypeptide N-acetylgalactosaminyl tranferase. Glycoconj. J., 12, 824–828.[ISI][Medline]

Yoshida, A., Suzuki, M., Ikenaga, H., and Takeuchi, M. (1997) Discovery of the shortest sequence motif for high level mucin-type O-glycosylation. J. Biol. Chem., 272, 16884–16888.[Abstract/Free Full Text]

Young, J.D., Tsuchiya, D., Sandlin, D.E., and Holroyde, M. (1979) Enzymic O-glycosylation of synthetic peptides from sequences in basic myelin protein. Biochemistry, 18, 4444–4448.[ISI][Medline]

Zara, J., Hagen, F.K., Ten Hagen, K.G., van Wuyckhuyse, B.C., and Tabak, L.A. (1996) Cloning and expression of mouse UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase-T3. Biochem. Biophys. Res. Commun., 228, 38–44.[CrossRef][ISI][Medline]

Zhang, Y., Wang, P.G., and Brew, K. (2001) Specificity and mechanism of metal ion activation in UDP-galactose:ß-galactoside-{alpha}-1, 3-galactosyltransferase. J. Biol. Chem., 276, 11567–11574.[Abstract/Free Full Text]

Zhang, Y., Iwasaki, H., Wang, H., Kudo, T., Kalka, T.B., Hennet, T., Kubota, T., Cheng, L., Inaba, N., Gotoh, M., and others. (2002) Cloning and characterization of a new human UDP-N-Acetyl-{alpha}-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase, designated pp-GalNAc-T13, that is Specifically expressed in Neurons and Synthesizes Tn Antigen. J. Biol. Chem., forthcoming.

Zheng, X. and Sadler, J.E. (2002) Mucin-like domain of enteropeptidase directs apical targeting in madin-darby canine kidney cells. J. Biol. Chem., 277, 6858–6863.[Abstract/Free Full Text]