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 (GalNAc1-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., 1998), sugar-nucleotide transporters (e.g., Oelmann et al., 2001
), and glycosyltransferases (Marth, 1996
; Muramatsu, 2000
; Furukawa et al., 2001
) 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, 1970
) 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 (GalNAc1-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., 1997
, 1998
) based on amino acid sequence similarities (see Coutinho and Henrissat, 1999
). Because O-linked glycosylation proceeds step-wise (Strous, 1979
), 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, 1990; Moody et al., 2001
; Xu and Weiss, 2002
). Structurefunction 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., 2000
]; apolipoprotein A [Garner et al., 2001
]). O-linked carbohydrate side chains function as ligands for receptors (e.g., in hostmicrobial interactions [Hooper and Gordon, 2001
]; lymphocyte and leukocyte homing [Yeh et al., 2001
; Somers et al., 2000
]) and as signals for protein sorting (Alfalah et al., 1999
; Altschuler et al., 2000
; Breuza et al., 2002
; Naim et al., 1999
; Zheng and Sadler, 2002
).
ppGaNTase activity was first reported some 35 years ago by McGuire and Roseman (1967), 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., 1982); it had an apparent molecular mass of 55,000 Da. Elhammer and Kornfeld (1986)
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., 1995
); 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., 2001 for general commentary about the relative overlap between the public and private genome databases).
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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), ppGaNTase-T10 and reserve "-T9" for the isoform described by Toba et al. (2000)
that has recently been shown to have enzymatic activity (Zheng et al., 2002
). Following this proposed scheme, the next isoform with demonstrated (published) activity should be assigned ppGaNTase-T11 (Schwientek et al., 2002
), then -T12 (Guo et al., 2002
) followed by -T13 (Zheng et al., 2002
), and so on.
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Conceptual translation of ppGaNTase cDNAs reveals that these enzymes are type II membrane proteins, characterized by a short (424 aa) N-terminal cytoplasmic tail, followed by a small (1525 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., 2001), but this has not yet been established for any of the ppGaNTases as yet. ppGaNTase-T5 (Ten Hagen et al., 1998
) 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., 1998
; Breton et al., 2001
).
Because ppGaNTases are found in external secretions such as colostrum (e.g., Hagen et al., 1993), it is likely that the stem region is susceptible to proteolytic cleavage via a membrane protein secretase (Hooper et al., 1997
). 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
2,6 galactose linkages (Kitazume et al., 2001
). 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) 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., 1995
) 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., 2001
). Similarly, structural studies of UDP-Gal:ß-galactoside
1,3 galactosyltransferase (Gastinel et al., 2001
; Boix et al., 2001
) 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., 2001
).
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, 2001). 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, 2001
). Either histidine or carboxylate acidcontaining 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, 2001
). 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., 1990
). 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) have proposed that ppGaNTases share a common structural fold containing a parallel ß-sheet flanked by
-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, 1998
) 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., 2000
). 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., 2002
).
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Structurefunction 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) 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, 2002
). 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., 1986). Significantly, the ppGaNTase family has been identified both in silico and biochemically in both C. elegans (Hagen et al., 2001
; Hagen and Nehrke, 1998
) and Drosophila (Ten Hagen and Tran, 2002
; Schwientek et al., 2002
).
Mika et al. (2001) recently analyzed phylogenetic trees for a number of glycosyltransferase families. They estimate a divergence of ß1,4galactosyltransferases from ppGaNTases some 12001500 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 earlybetween 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) 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., 1999a
; Schwientek et al., 2002
; Guo et al., 2002
). Interestingly, Drosophila T1 (Drosophila pgant35A; Ten Hagen and Tran, 2002
) 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., 2002
). 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., 2000 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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The Clausen group has prepared a series of monoclonal antibodies against human ppGaNTase-T1, -T2, -T3, -T4, -T6, and -T11 (Bennett et al., 1998, 1999a
; Mandel et al., 1999
; Schwientek et al., 2002
). These reagents could prove valuable in delineating patterns of isoform-specific expression in tissues, organs, and cells. For example, Schwientek et al. (2002)
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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Enzyme activity and specificity
Only a subset of hydroxyamino acids is substituted with GalNAc in the mammalian proteome (Apweiler et al., 1999). 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., 1979; Hughes et al., 1988
; Briand et al., 1981
; Cottrell et al., 1992
). The octapeptide VTPRTPPP was determined to be the best of these substrates, and evidence suggested that both threonine residues were glycosylated (Cottrell et al., 1992
). 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., 1994a
,b
; Stadie et al., 1995
), MUC2 (Tetaert et al., 1994
; Inoue et al., 1998
, 2001
; Iida et al., 2000
; Kato et al., 2001a
,b
), and the respiratory mucin MUC5AC (Hennebicq et al., 1998a
,b
) 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. (1996, 1997
) 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)
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., 1996
, 1997
). 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., 1996
). One possible explanation is that a novel ppGaNTase is induced by the temperature shift (see Henle et al., 1990
). 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) 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, 1994). Recent technical advances in mapping O-glycosylation, including the use of Edman degradation (Gooley and Williams, 1994
; Gerken et al., 1997
) and mass spectrophotometric methods following various modes of chemical derivatization (for review, see Hanisch et al., 2001a
; Mirgorodskaya et al., 2001
; Czeszak et al., 2002
, and references therein), have made it possible to map native multisite substrates.
The original analyses of O'Connell et al. (1991) and Wilson et al. (1991)
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)
. 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., 1991
; Wilson et al., 1991
), aspartic acid and glutamic acid are found often around multiple glycosylation sites (Christlet and Veluraja, 2001
). Elhammer et al. (1993)
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 (1994a
,b
).
Several neural network approaches have been explored to predict O-glycosylation sites (Cai et al., 1997, 2002
; Hansen et al., 1998
). 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., 1998
). 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)
demonstrated that several reporter substrates predicted not to be glycosylated by NetOGlyc do acquire O-glycans in specific cell backgrounds. Gerken et al. (1997)
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., 1998
).
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., 1998). 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., 1992
, 1993
), human von Willebrand factor (O'Connell et al., 1992
) and erythropoietin (O'Connell et al., 1992
; Wang et al., 1993
; Yoshida et al., 1997
; Elhammer et al., 1999
) have been used to challenge the ppGaNTase isoforms. Results obtained corroborate earlier findings with tissue extractsflanking 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., 1999
). 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., 1997
, 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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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., 2001). 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)
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) suggested that the lack of secondary structure leads to increased accessibility to a partially purified ppGaNTase, most likely ppGaNTase-T1. Nishimori et al. (1994a)
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., 1993
) does not affect the acquisition of O-linked GalNAc. O'Connell et al. (1991)
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)
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., 1997
) 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., 1994). 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., 1990, 1996
) 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)
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., 1999
). 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., 2001b
).
Bennett et al. (1998) 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
-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., 1997
) and was subsequently shown to add GalNAc to a variety of other peptide substrates (Bennett et al., 1999a
) (summarized in Table IV), this isoform could use a glycopeptide substrate as well.
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Presently there appear to be two types of glycopeptide ppGaNTasesthose 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., 1997, 1998
, 2002
). They find that the core 1 structure Galß1,3GalNAc
is more likely to occur in less dense regions of O-glycosylation. In contrast, the addition of
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) demonstrated by quantitative analysis of labeling with a monoclonal antibody with specificity for
-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., 1994
). 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)
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., 2001
), 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) ablated expression of a highly homologous form of ppGaNTase-T1 (which is now designated ppGaNTase-T13; Zhang et al., 2002
). 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., 1999
). 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), Erickson (1993)
, and Pearson (2002)
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)
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, 2002
). 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 (PMT17). Gene disruption of two or more pmt genes encoding the O-mannosyltransferases is required before a resultant growth defect is observed (Gentzsch and Tanner, 1996
). 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, 2002
; Schwientek et al., 2002
). 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., 1999, 2001
; Tomana et al., 1999
; Iwase et al., 2001
). There is no evidence of any nucleotide sequence alteration or transcriptional change in the
1 hinge region (Greer et al., 1998
). Rather, the alteration is thought to be posttranslational in nature. Mattu et al. (1998)
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)
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., 2002
).
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, 1990). O-glycans also serve as the recognition site of many ligands including selectins (Yeh 2001
). Hakomori (2002)
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, 1995
). 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., 2001
). Horan et al. (1999)
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., 1986), unless the cells are cultivated under conditions that allow O-glycosylation to proceed uninhibited. Subsequently, Davis et al. (1986)
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., 1991
). 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., 1991
). 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.
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Footnotes |
---|
1 To whom correspondence should be addressed; e-mail: Lawrence.Tabak{at}nih.gov
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.
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