2Department of Anatomy and Cell Biology, 1600 SW Archer Road, University of Florida College of Medicine, Gainesville, FL 32610-0235, USA, and 3Department of Chemistry, University of Florida, Gainesville, FL 32611-7200, USA
Accepted on November 2, 2001;
Abstract
Recently, complex O-glycosylation of the cytoplasmic/nuclear protein Skp1 has been characterized in the eukaryotic microorganism Dictyostelium. Skp1s glycosylation is mediated by the sequential action of a prolyl hydroxylase and five conventional sugar nucleotidedependent glycosyltransferase activities that reside in the cytoplasm rather than the secretory compartment. The Skp1-HyPro GlcNAcTransferase, which adds the first sugar, appears to be related to a lineage of enzymes that originated in the prokaryotic cytoplasm and initiates mucin-type O-linked glycosylation in the lumen of the eukaryotic Golgi apparatus. GlcNAc is extended by a bifunctional glycosyltransferase that mediates the ordered addition of ß1,3-linked Gal and 1,2-linked Fuc. The architecture of this enzyme resembles that of certain two-domain prokaryotic glycosyltransferases. The catalytic domains are related to those of a large family of prokaryotic and eukaryotic, cytoplasmic, membrane-bound, inverting glycosyltransferases that modify glycolipids and polysaccharides prior to their translocation across membranes toward the secretory pathway or the cell exterior. The existence of these enzymes in the eukaryotic cytoplasm away from membranes and their ability to modify protein acceptors expose a new set of cytoplasmic and nuclear proteins to potential prolyl hydroxylation and complex O-linked glycosylation.
Key words: glycosyltransferase evolution/cytoplasmic glycosylation/Dictyostelium/prolyl hydroxylase/Skp1
Introduction
The secretory pathway has long been considered the provenance of protein glycosylation in eukaryotes, so it has always been interesting when evidence is presented for glycosylation of cytoplasmic or nuclear proteins. This review will focus on complex glycosylation, defined by the addition of a chain of at least two sugars to a protein amino acid, because "simple" modification of Ser/Thr residues by O-ß-GlcNAc has been recently reviewed (Thornton et al., 1999; Comer and Hart, 2000
; Wells et al., 2001
). As concluded by a comprehensive summary in 1989 (Hart et al., 1989
) and updated in 1999 (in Varki et al., 1999
), considerable data support the existence of cytoplasmic/nuclear glycoproteins with complex glycans. At the cellular level, immunocytochemical studies based on lectins, anticarbohydrate antibodies, and glycosaminoglycan-binding proteins localize glycoconjugates to the cytoplasm and nucleus. At the molecular level, cytoplasmic/nucleoplasmic proteins can be labeled with lectins in periodate-sensitive fashion, metabolically labeled with radioactive sugar precursors, or shown to contain chemical quantities of sugars after purification. However, as also summarized in those reviews, direct structural studies are required to support these interpretations. For example, recent work failed to confirm glycosylation of large T antigen and a high mobility group protein except for modification by O-ß-GlcNAc (Medina and Haltiwanger, 1998a
,b). In parallel, the discovery of extracellular functions for some cytoplasmic/nuclear lectins (Hughes, 2001
; Lowe, 2001
) has diminished the likelihood that intracellular glycan ligands exist. Nevertheless, complex cytoplasmic glycosyation does occur, as reviewed here, and so questions that relate to its prevalence and significance to the cell are relevant.
Evidence for cytoplasmic/nuclear proteins with complex glycans
A compilation of eukaryotic cytoplasmic or nuclear proteins that are known or suspected to be glycosylated is given in Table I. A compelling case has been presented for complex glycosylation of Paramecium bursaria Chlorella virus-1 (PBCV-1) capsid proteins in the cytoplasm of Chlorella algae. This virus is assembled in the cytoplasm away from membranes, consistent with the lytic mode of viral dispersion (Van Etten et al., 1991). Its VP54 capsid protein is metabolically labeled with [3H]Gal and contains Fuc, Gal, Man, Xyl, Ara or Rha, and Glc (Wang et al., 1993
), and its Mr is shifted from 54,000 to 49,000 by chemical deglycosylation. Six spontaneous virus mutants, isolated in a screen for resistance to an anticarbohydrate virus antibody, express Mr variants of VP54 that correlate with altered sugar composition, providing strong evidence for a covalent association of sugars with the protein (Que et al., 1994
). These results led to a proposed glycan structure and a corresponding biosynthetic pathway of six virally encoded glycosyltransferases (GTases) that reside in the cytoplasm. BLAST analysis of the PBVC-1 genome yields six candidate GTase genes. Most of these lack known targeting motifs for the secretory pathway, and one exhibits remote similarity to "Fringe"-type GTases and contributes to VP54 glycosylation based on genetic analyses (Graves et al., 2001
).
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A potential example of dynamic cytoplasmic glycosylation is that of the phosphoglucomutase-family protein parafusin (Levin et al., 1999). Phosphorylation and membrane association of parafusin oscillates with secretory activity of the cell. Phosphate is proposed to be incorporated in crude extracts into a Glc
1-PO4-Man moiety on an unknown O-linked glycan, based on indirect product characterization from radioactive precursors (Satir et al., 1990
; Marchase et al., 1990
, 1993; Veyna et al., 1994
). Partial corroborating evidence was obtained for parafusin metabolically labeled with [2-3H]Man, but interpretation of the phosphosugar linkages is confounded by recent reports of extensive Ser-phosphorylation (Kussman et al., 1999
) that was not originally reported. If this model can be supported by direct structural studies, parafusin has the potential to greatly expand our perspective on dynamic cytoplasmic glycosylation.
Plant nuclear pores contain proteins that are recognized by wheat germ agglutinin and can be radiolabeled by UDP-[3H]Gal and soluble ß1,4-Gal-Tase, indicating terminal GlcNAc residues (Heese-Peck et al., 1996). Unlike animal nuclear core complex proteins modified by clustered, single O-ß-GlcNAc residues, alkaline degradation released products approximately five sugars in length, with no evidence for a peptide based on enzymatic and chemical tests. These proteins might be modified by a glycan(s) with a nonreducing terminal GlcNAc, O-linked to Ser or Thr, but proof awaits direct demonstration of the structure.
Another report suggested that cytokeratins are modified by a GalNAc1,3-terminated oligosaccharide(s), based on lectin binding and compositional analysis of the purified protein (Goletz et al., 1997
). Binding was diminished by pretreatment with
-N-acetyl-galactosaminidase and did not occur with recombinant cytokeratin. The implication that cytokeratins might be ligands for endogenous galectin(s) is intriguing but awaits direct structural evidence to rule out the possibility of a contaminating glycoprotein.
Although it is a special case, the primer for glycogen synthesis, glycogenin, was the first-known cytoplasmic glycoprotein (Alonso et al., 1995). A Tyr-residue on glycogenin serves as the attachment site for a sequentially added
1,4-linked Glc oligomer that is applied by yet another glycogenin protein doubling as a GlcTase (Lin et al., 1999
).
Examples of N-linked glycans in the cytoplasm probably originate in the secretory pathway. There is good evidence for the N-glycosylation of a mitochondrial protein, but it appears to be imported from the rough endoplasmic reticulum (RER) (Chandra et al., 1998). An N-glycosylated nuclear lectin has been also found in the RER and Golgi (Rousseau et al., 2000
). It is now also appreciated that unfolded RER glycoproteins are targeted to the cytoplasm for degradation (Cacan and Verbert, 2000
), but their sugar chains are presumably transient and nonfunctional in this compartment. If evidence can be obtained that N-linked glycans associated with tau protein from paired helical filaments of Alzheimers brains (Sato et al., 2001
) is not due to contaminant proteins, it would suggest a breakdown in compartmentalization in a pathological situation. Overall, the observations raise the possiblity of N-glycan function in the cytoplasm or nucleus.
It is worth noting that the bulk of eukaryotic cytoplasmic glycosylation probably occurs on exported polysaccharides, including hyaluronan (DeAngelis, 1999), chitin, cellulose (Delmer, 1999
), other glucans, and lipids that are subsequently flipped across membranes as precursors for protein N-glycosylation (Schenk et al., 2001
; Burda and Aebi, 1999
), glycophosphatidylinositol anchors (Tiede et al., 1999
), and glycosphingolipids initiated with Glc (Marks et al., 1999
). Some glycolipids, such as sterol glucosides or mannosides, appear to remain oriented toward the cytoplasm (Warnecke et al., 1999
). A polysaccharide that is normally exported, hyaluronan, also appears to accumulate in the cytoplasm and nucleus based on immunocytochemical studies and evidence for natural cytoplasmic and nuclear receptors (Lee and Spicer, 2000
; Huang et al., 2000
). Intracellular hyaluronan might result from failure to translocate to the cell exterior. Older biochemical and microscopic evidence also exists for the accumulation of proteoglycans or derived glycosaminoglycans (including heparan, chondroitin, and dermatan sulfates) in the cytoplasm and nucleus of brain and cultured mammalian cells, which was suggested to involve endocytosis and processing of conventionally secreted proteoglycans (Fedarko et al., 1989
; Busch et al., 1992
; Margolis and Margolis, 1993
; Hiscock et al., 1994
).
Complex cytoplasmic glycosylation of Skp1 in Dictyostelium
The best studied example of a cytoplasmic/nuclear glycoprotein and a corresponding cytoplasmic glycosylation pathway has emerged from the analysis of Skp1 in the "lower" eukaryote Dictyostelium, known best for cell biological studies of development. Dictyostelium has also been an attractive model organism for glycobiology, because it possesses an N-glycosylation pathway lacking typical complex processing, with some novel variations, and several Golgi-associated O-linked pathways whose products, including phosphoglycosylation, are immunogenic (Freeze, 1997; in Varki et al., 1999
). The International Dictyostelium DNA sequencing efforts have nearly saturated the genome, and laboratory strains of this haploid organism are well suited for biochemical analysis, in addition to knockout and knockin genetic manipulations and restriction enzymemediated insertional mutagenesis screens to investigate function (Sucgang et al., 2000
).
Skp1 is a small (Mr = 21,000), rather remarkable protein found in several multiprotein complexes of the cytoplasm and nucleus of yeast, plants, invertebrates, and vertebrates. Best characterized is the SCF (Skp1, cullin-1, F-box protein, Roc1/Rbx1) subunit of the E3 ubiquitin ligase complex that targets specific phosphoproteins, including cell cycle regulatory proteins and transcriptional factors, for polyubiquitination and subsequent degradation in proteasomes (Deshaies, 1999). Phosphoprotein target specificity is mediated by the F-box protein subunit, represented by 35 different gene products in mammals. The F-box domain contacts Skp1, which in turn interacts with cullin-1 and an E2 ubiquitin-conjugating enzyme. Recent evidence suggests that SCF can alternatively bind Snf1-related protein kinases and proteasomes (Farras et al., 2001
). Skp1 also appears to occur in non-SCF complexes, including the centrosomal CBF3 complex, a kinetochore complex, the RAVE-complex that might regulate assembly of the vacuolar proton transporter, and another complex(es) possibly involved in membrane trafficking (Seol et al., 2001
), altogether suggesting a special role for Skp1 at the junction of multiple intracellular regulatory pathways.
Glycosylation of Skp1 was originally detected in Dictyostelium by metabolic incorporation of [3H]Fuc (Gonzalez-Yanes et al., 1992), and subsequently confirmed by compositional analysis of the purified protein showing the presence of GlcNAc, Fuc, Gal, and possibly Xyl (Kozarov et al., 1995
; Teng-umnuay et al., 1998
). Purification of radioactivity from [3H]Fuc-labeled Skp1 peptides showed only a single site of incorporation of Fuc into the protein (Teng-umnuay et al., 1998
). Tandem mass spectrometry (MS) and Edman sequencing showed that the glycopeptide contains a pentasaccharide attached to a HyPro residue at position 143 (Figure 1). Based on exoglycosidase digestions, the core trisaccharide has the structure of the type 1 blood group H antigen and is modified by two
-linked Gals. Coordinate loss of the outer three sugars during mild acid hydrolysis suggested linkage of the outer Gals to Fuc, consistent with the absence of significant
-galactosylation in a GDP-Fuc-deficient mutant, but this has not been determined directly.
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The great majority of Skp1 in both growing and developing Dictyostelium cells appears to be fully glycosylated based on western blot Mr analysis of whole cell Skp1 (Sassi et al., 2001). Matrix-assisted laser desorption and ionization time-of-flight MS analysis of peptides from purified Skp1 supports this conclusion but does also detect unglycosylated, unhydroxylated peptide (Teng-umnuay et al., 1998
). Evidence therefore suggests that Skp1 glycosylation is nearly stoichiometric.
The possibility that Skp1 is similarly glycosylated in other organisms remains largely unexplored. Preliminary sugar composition studies show that highly overexpressed Skp1 in Saccharomyces cerevisiae is not significantly glycosylated, and treatment of cultured mammalian cells with prolyl 4-hydroxylase inhibitors fails to shift their apparent Mr values (West et al., unpublished data). The equivalent of Pro143 occurs in microbial, plant, and invertebrate Skp1s, but not in vertebrate homologs, though a related Pro residue is found nearby (West et al., 1997). It is difficult to draw general conclusions from these observations, however, because protein O-glycosylation is notoriously polymorphic both phylogenetically and developmentally. Direct structural studies on other Skp1s awaits purification of native material from other organisms. A search for other fucoproteins in the cytosolic fraction of Dictyostelium has failed to yield other candidates based on either metabolic labeling or in vitro fucosylation of extracts from fucosylation deficient cells (West et al., 1996
). However, these results do not rule out membrane-associated, developmentally regulated or low-abundance fucoproteins.
Potential functions of Skp1 glycosylation
As cited above, Skp1 participates in a newly emerging network of intracellular regulatory pathways. Consistent with multiple functions, Skp1 localizes to several subcellular sites (Freed et al., 1999; Gstaiger et al., 1999; Sassi et al., 2001). Biochemically, soluble Skp1 purifies into major and minor pools (West et al., 1997
) that appear to differ in the content of a single Gal residue, and a fraction of Skp1 is associated in a salt-sensitive fashion with the microsomal fraction in Dictyostelium (Kozarov et al., 1995
). Multiple genetic isoforms of Skp1 exist in most eukaryotes in addition to potential glycoforms. In Dictyostelium, there is no evidence for a correlation of either of the two genetic isoforms with subcellular location (Sassi et al., 2001
) or biochemical fractionation (West et al., 1997
), and the great majority (>90%) of these Skp1s are normally glycosylated. However, these results do not exclude the possibility that specific Skp1 functions are mediated by structural variants.
The best available clue for why Skp1 is glycosylated is that when it is inhibited mutationally or pharmacologically, Skp1 is no longer concentrated in the nucleus (Sassi et al., 2001). Mutant analysis shows that the core Galß1,3GlcNAc disaccharide is sufficient for this function. Proteasomes are enriched in the nucleus, suggesting that nuclear Skp1 might belong to the SCF complex. If the SCF complex is assembled in the cytoplasm, Skp1 might enable its nuclear targeting. Consistent with such a role, the position of the Skp1 pentasaccharide near its C-terminus appears to be oriented away from known contacts with other proteins in the SCF complex (Schulman et al., 2000
). However, the effect of glycosylation may be indirect, because the ability of Skp1 to target a Skp1/GFP hybrid to the Dictyostelium nucleus does not depend on glycosylation (Erogul, 2001
). Nonglycosylated Skp1 can accumulate to normal levels and is stable when cells dedifferentiate (Sassi et al., 2001
), as if glycosylation is not required for stability.
The addition of specific sugars is inhibited in Skp1s expressed with point mutations elsewhere in the protein (Sassi et al., 2001), raising the possibility that glycosylation depends on normal Skp1 structure. Though this might simply reflect the inherent specificity of the modification enzymes, it is intriguing to consider these results from a quality control perspective. Roles in quality control of folding have been advanced for collagen prolyl 4-hydroxylation (Lamande and Bateman, 1999
) and N-linked glycan processing of proteins in the RER (Helenius and Aebi, 2001
). If glycosylation is required to certify that Skp1 has folded and assembled into multiprotein complexes properly, this could explain why unglycosylated Skp1 is unable to accumulate in the nucleus.
Another potential role for Skp1 modification is as a metabolic sensor that depends on adequate supply of cofactors and substrates for the modification enzymes. For example, physiologic O2 availability appears to be rate-limiting for the prolyl 4-hydroxylation of a hypoxia-sensitive transcriptional factor in mammals (hypoxia-inducing factor-), thereby regulating its ubiquitinylation by the SCF-like von Hippel-Lindau complex (Ivan et al., 2001
; Jaakkola et al., 2001
). The level of O-ß-GlcNAc modification of cytoplasmic/nuclear proteins has been suggested to depend on the availability of Glc and GlcNAc over physiological ranges (Akimoto et al., 2001
). Fucosylation of Skp1 in vitro is highly sensitive to inhibition by nucleotides (West et al., 1996
). Reduced O2 availability might explain why Skp1 expressed during multicellular development is not glycosylated (Sassi et al., 2001
).
Mechanism of Skp1 glycosylation
Skp1 glycosylation is expected to require a prolyl hydroxylase and five GTases (Figure 1). The enzyme activities have each been assayed using various mutant and recombinant Skp1s that are incompletely hydroxylated or glycosylated in vivo as acceptor substrates. Based on these assays, Skp1 appears to be modified sequentially by a pathway of soluble enzymes in the cytoplasmic rather than the secretory compartment of the cell. Glycosylation can occur posttranslationally in vivo, as Skp1 produced in prespore cells becomes glycosylated when cells are dedifferentiated into vegetative cells (Sassi et al., 2001). Overexpressed Skp1 is efficiently glycosylated, suggesting a high capacity for the pathway consistent with the limited glycosylation microheterogeneity of normal Skp1.
Prolyl hydroxylase
The mechanism of prolyl hydroxylation appears to be related to that of collagen, based on the ability of low concentrations of known inhibitors of prolyl 4-hydroxylases to cause a downward shift in Mr similar to that caused by mutation of the Pro143 attachment site to an Ala residue (Sassi et al., 2001). A search of Dictyostelium cDNA and genomic DNA sequence databases reveals a putative gene (gene f01884; http://dicty.sdsc.edu/annotationdicty.html) similar to the prolyl 4-hydroxylase encoded by the above-mentioned Chlorella viral genome (Eriksson et al., 1999
). The PBCV-1 prolyl 4-hydroxylase contains a canonical catalytic domain but lacks the N-terminal domain thought to interact with protein disulfide isomerase in the RER. Dictyostelium gene f01884 contains all five of the key conserved residues considered to be diagnostic for these enzymes but, in contrast to known prolyl 4-hydroxylases, lacks sequences for targeting its product to the RER, where prolyl 4-hydroxylases usually reside (Kivirikko and Pihlajaniemi, 1998
). Hydroxylation of Skp1 Pro143 is thus suggested to occur by a conventional posttranslational mechanism using a prolyl 4-hydroxylase that is novel both in its compartmentalization and in its lack of a requirement for repeat motifs typically found in prolyl 4-hydroxylase substrates.
GlcNAcTase
The Skp1 GlcNAcTase was assayed by measuring transfer [3H] of from UDP-[3H]GlcNAc to a mutant Skp1 deficient in GlcNAc (Teng-umnuay et al., 1999). A synthetic peptide containing 4-HyPro143 was a moderately competitive substrate, whereas nonhydroxylated forms of Skp1 or peptide were not. Little or no transferase activity was detected in microsomal fractions, suggesting the enzyme is cytosolic. The GlcNAcTase activity is dependent on a divalent cation and reducing conditions, and exhibits submicromolar Kms for its substrates, properties expected to support efficient function in the cytoplasm. Purification of the Skp1 GlcNAcTase activity was facilitated by the construction of a novel affinity resin linking UDP-GlcNAc at its 5-uridyl position that might be applicable to other enzymes of this class. Activity was associated with a single Mr 51,000 protein (GnT51), after 130,000-fold purification to near homogeneity, which contains the binding site for UDP-GlcNAc based on photoaffinity labeling.
Peptide sequences obtained from tryptic digests of GnT51 were sequenced de novo on a hybrid quadrupole time-of-flight (Q-TOF) tandem MS instrument (West et al., 2001), which was well-suited for the limited amount of protein available from the purification. These sequences made possible a polymerase chain reactionbased approach to identify partial GnT51 genomic sequence which was extended with sequences from the International Dictyostelium genome project. This yielded a predicted 2-exon ORF that encodes a protein with an Mr of 52,000 similar to that obtained by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and contains many of the GnT51 peptides characterized by Q-TOF MS. Expression of the predicted cDNA in Escherichia coli resulted in Skp1 GlcNAcTase activity in the soluble cell extracts. The N-terminus of the predicted sequence lacks motifs for targeting to the RER, consistent with the biochemical evidence that this is a cytoplasmic enzyme.
The N-terminal 260 amino acids of GnT51 display sequence similarity to the catalytic domain of mucin-type UDP-GalNAc:Ser/Thr polypeptide (PP) GalNAcTases of family 27 (Campbell et al., 1997
), though this was too weak to be detected by BLAST algorithms (Altschul et al., 1990
, 1997). Greatest similarity lies in two regions: the N-terminal 125 amino acids known as the NRD2-subdomain (Kapitonov and Yu, 1999
), which includes a DxH sequence related to the commonly occurring DxD motif, and downstream in the previously recognized Gal/GalNAc box (Hagen et al., 1999
), a sequence of about 60 amino acids in what we call the cat27 domain. The alignment in Figure 2A highlights the regions of greatest similarity, and is shaded to emphasize the chemical relatedness of amino acid positions in light of the divergence of specific amino acid identities. Sequences from more distantly related families are compared to illustrate the significance of the family 27 relatedness. A phylogenetic analysis of the sequences supports the close relationship of GnT51 to family 27 (Figure 3, green clade). Family 7 ß4GalTases and certain putative family 2 GTases with unknown function are also found near the base of this clade, but GnT51 appears to be more related to family 27 sequences based on the greater motif similarity seen in the Figure 2A alignment and the presence of a short C-terminal domain that recognizes acceptor substrates in some family 27 enzymes (Hassan et al., 2000
). Animal PP GalNAcTases catalyze formation of GalNAc
1-Ser/Thr linkages, which are related to the GlcNAc-HyPro linkage formed by the Skp1 GlcNAcTase. By analogy, the GlcNAc of Skp1 is predicted to be
-linked, in contrast to the ß-linkage of GlcNAc catalyzed by the unrelated cytoplasmic/nuclear Ser/Thr O-ß-GlcNAcTase (Comer and Hart, 2000
).
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ß1,3Galactosyltransferase and 1,2fucosyltransferase
The second and third sugars of the Skp1 glycan are added by a single protein, FT85, with two GTase domains. FT85 was originally purified from the cytosolic fraction as the FucTase, which was assayed as transfer of [3H] from GDP-[3H]Fuc to Skp1(modC) from a GDP-Fuc synthesis mutant, resulting in formation of a Fuc1,2Gal linkage (West et al., 1996
; Trinchera and Bozzarro, 1996
). Little or no activity was detectable in the microsomal fraction, which was not due to an inhibitor based on mixing studies. Purification over 1 millionfold led to the identification of an Mr 85,000 protein, FT85, which copurified with the activity and was photoaffinity-labeled with GDP-hexanolamine-[125I]ASA (West et al., 1996
). Like the Skp1-GlcNAcTase, the FucTase also has properties of a cytoplasmic enzyme in its requirement for a reducing agent, its neutral pH optimum, and submicromolar Kms for its substrates. It is remarkably sensitive to cytochrome c and GDP in vitro, the significance of which is not known.
FT85 can also fucosylate the acceptor disaccharide Galß1,3GlcNAc when attached to a hydrophobic aglycon, albeit with a Km three orders of magnitude higher (West et al., 1996). Galß1,4GlcNAc (type 2) and Galß1,6GlcNAc (type 3) moieties are much poorer acceptors. GalNAc can substitute for GlcNAc and may be either
- or ß-linked to the aglycone. The substrate preferences of the enzyme most resembles that of the mammalian Se-type
1,2-FucTase, but, in contrast to the Golgi enzyme, the Skp1 FucTase is absolutely dependent on a divalent cation such as Mg2+ for activity.
The proteomics strategy used to clone the GnT51 gene was also successfully applied to FT85 (Van der Wel et al., 2001a), yielding a predicted coding region of 768 amino acids. FT85 is required for fucosylation of Skp1 in vivo as determined by targeted disruption of its gene locus. In addition, expression of FT85 in E. coli rendered soluble extracts capable of fucosylating highly purified Skp1. Consistent with the biochemical data, FT85 lacks known motifs for targeting to the RER or for membrane association. Thus FT85 is necessary and sufficient for Skp1 FucTase function in the cytoplasm (or nucleus) of the cell.
Skp1(FT85) from the FT85-null strain was fortuitously discovered to be a good substrate for a UDP-Gal-dependent GalTase activity in the crude cytosolic extract (Van der Wel et al., 2001b). MS structural studies carried out as described previously (Teng-umnuay et al., 1998
) showed that the Skp1(FT85) glycopeptide contains only the reducing terminal GlcNAc attached to HyPro. FT85-null extracts are devoid of Skp1 ßGalTase activity, and highly purified Dictyostelium FT85 and recombinant FT85, exhibit abundant activity. The reaction product is susceptible to ß1,3-galactosidase digestion. Thus FT85 is a bifunctional glycosyltransferase.
The sequence of FT85 harbors two GTase-like domains, each about 250 amino acids, that are characteristic of bacterial family 2 inverting GTases (Campbell et al., 1997). As shown by the sequence alignment in Figure 2B, the putative domains each contain a variation of the NRD2-subdomain described above for the Skp1 GlcNAcTase, including characteristic Asp residues and a DxD motif (highlighted in magenta) critical for coordinating the sugar nucleotide donor (Charnock and Davies, 1999
; Garinot-Schneider et al., 2000
). The second half of each putative domain contains sequences associated with the predicted catalytic base of family 2 domains (highlighted in magenta). This region is highly divergent between family 2 and family 27 enzymes correlating with inversion and retention, respectively, of the configuration of the sugar linkages formed. The alignments also include more distantly related sequences from family 2 and other families for comparison. The family 2 catalytic domain has recently been recognized as belonging to a GTase superfamily that includes the eukaryotic Golgi GTases GlcNAcTase-I (initiates complex N-glycan processing), ß1,4GalTase,
1,3GalTase, and a GlcUATase based on structural homologies (Unligil and Rini, 2000
; Pederson et al., 2000
; Gastinel et al., 2001
; Persson et al., 2001
).
Recombinant expression of the FT85 N- and C-terminal domains in FT85-mutant cells shows that they mediate the Skp1 ßGalTase and FucTase activities, respectively, confirming the two-domain model predicted by the sequence analysis. Amino acid substitutions in these domains specifically diminish the corresponding activities and support the family 2 classification (Van der Wel et al., unpublished data). A phylogenetic analysis of the sequences shows that the ß1,3GalTase domain is more related to a bacterial (Figure 3) than known eukaryotic ß1,3GalTases, which could not be coaligned. The 1,2FucTase C-terminal domain is found in a clade enriched in eukaryotic GTase or GTase-like sequences and shows no resemblance to previously described
1,2FucTases (Oriol et al., 1999
). The FT85 GTase domain sequences are intermingled among evolutionary lineages that have produced multiple modern prokaryotic and eukaryotic GTases, which are themselves intermingled (albeit with low bootstrap support; Figure 3). These observations suggest that the eukaryotic family 2 GTases evolved multiple times, possibly along lineages corresponding to catalytic specificity rather than membrane anchorage or the transition from prokaryotes to eukaryotes.
The two-domain architecture of FT85 resembles that of bifunctional prokaryotic GTases involved in the synthesis of hyaluronan (Jing and DeAngelis, 2000), chondroitin (DeAngelis and Padgett-McCue, 2000
), and other bacterial polysaccharides. For example, the class II hyaluronan synthase consists of two family 2 domains that catalyze formation of the alternating ß1,3GlcUA- and ß1,4GlcNAc-linkages of hyaluronan, based on mutagenesis studies. Bifunctional GTases have also been implicated in the formation of terminal KDO-KDO and NeuAc-NeuAc linkages in bacteria (e.g., Belunis and Raetz, 1992
; Gilbert et al., 2000
), cellulose (Saxena and Brown, 2000
), heparan by EXT1 or EXT2 in the mammalian Golgi (Senay et al., 2000
), and animal hyaluronan by class I hyaluronan synthase (Yoshida et al., 2000
). FT85 is the first documented example of a two-domain bifunctional GTase in a eukaryote and is the first known to modify a protein substrate. This might ensure processive extension of the Skp1 glycan in the cytoplasm, where spatial control and compartmental protection as in the RER and Golgi are not available.
Other prospective cytoplasmic GTases
The compartmentalization of family 2 GTases in the cytoplasm was previously noted by Kapitonov and Yu (1999). In prokaryotes, the family 2 GTase domain is found in enzymes at the cytoplasmic face of the plasma membrane, where they contribute to the synthesis of polysaccharides and lipid-linked precursors of capsules and exopolysaccharides prior to their export to the cell surface (Whitfield and Roberts, 1999
). A topologically similar function is employed in eukaryotes for the synthesis of cellulose, chitin, hyaluronan, and other polysaccharides at the plasma membrane; Dol-P-Man and Dol-P-Glc at the cytoplasmic surface of the RER; and Glc-Cer at the Golgi surface. FT85 conserves the cytoplasmic localization of family 2 GTase domains, except that it is liberated from the membrane surface and extends the range of family 2 GTase acceptor substrates to include proteins.
The evolution of the Skp1 GlcNAcTase represents a similar scenario with the proviso that some of its closest known relatives are the mucin-type polypeptide GalNAcTases of the Golgi. The phylogenetic analysis suggests that these type II membrane proteins have evolved multiple times by the simple appending of an N-terminal signal anchor and adjacent stalk region. This model may also apply to a recently identified candidate GTase for cytoplasmic glycosylation of PBCV-1 viral capsid VP54 (Graves et al., 2001
), as its predicted sequence is most similar to Golgi GTases from families 31 and 34 (Campbell et al., 1997
).
Based on the Skp1 GTase studies, proteins containing family 2 and 27 catalytic domain-like sequences, themselves related, might contribute to the putative glycosylation of other cytoplasmic proteins summarized above and listed in Table I. A BLAST search in GenBank and other databases for new eukaryotic family 2 and 27like sequences that lack RER targeting sequences and are not apparently related to known subclasses that modify lipid or polysaccharide targets (e.g., Dol-P-Hex synthases or containing the processivity motif QXXRW), has yielded candidate open reading frames containing family 2 catalytic domains in the genomes of C. elegans, Arabidopsis thaliana, PBCV-1, and D. discoideum (Figure 2B). The absence of any candidate soluble family 2 GTases from vertebrates is notable but may be due to sequence divergence resulting in the failure of BLAST programs to identify homologous sequences. A phylogenetic analysis of these putative eukaryotic family 2 cytoplasmic enzymes suggests that they evolved multiple times from prokaryotes (Figure 3). For example, they are more related to other prokaryotic GTase genes than to each other, and they appear to have diverged prior to the evolution of the family 2 Dol-P-Hex synthases and the related family 23 ceramide GlcTases based on their distinct clading. These predicted enzymes appear to have remained in the cytoplasm when eukaryotes evolved from prokaryotes.
Two additional GTase families, out of the 55 total recognized by Henrissat (Campbell et al., 1997), contain cytoplasmic members that glycosylate eukaryotic polysaccharides and lipids. For example, glycogenin, the GlcTase that primes glycogen synthesis (Lin et al., 1999
), is classified in family 8 with other prokaryotic and eukaryotic presumptive GTases of unknown function, and these are candidates for mediating cytoplasmic glycosylation steps that retain the sugar linkage, based on the precedent of the family 2 member FT85. A similar argument applies to family 4, whose eukaryotic members include PigA, which forms the GlcNH2-inositol linkage in glycophosphatidylinositol-anchor synthesis (Tiede et al., 1999
); sucrose synthase; and a plant GalTase that forms digalactosyldiacylglycerol (Froehlich et al., 2001
), each of which is oriented toward the cytoplasm.
The frequent addition of O-ß-GlcNAc to Ser or Thr residues of animal cytoplasmic and nuclear proteins is mediated by a single, novel, soluble, metal-independent family 41 GTase (reviewed in Comer and Hart, 2000). A related enzyme is expressed in plants (Thornton et al., 1999
) and probably Dictyostelium. An unrelated O-ß-GlcNAcTase is secreted from Clostridium novyi and enters eukaryotic target cells to modify Rho GTP-binding proteins (Busch et al., 1998
). Unlike the
-linked GlcNAc of Skp1, there is as yet no evidence for the extension of O-ßlinked GlcNAc, but future studies might implicate these enzymes for initiating complex glycosylation of cytoplasmic proteins.
Context of complex cytoplasmic protein glycosylation
Although complex protein-linked glycans are certainly less prominent than O-ß-GlcNAc, their potential significance is enhanced by the novel functions tentatively associated with their distinctive sugar composition and linkages. Gal- and GalNAc-containing glycans might associate with galectins or discoidins that are prevalent in the cytoplasm (Cooper and Barondes, 1999) and nucleus (Vyakarnam et al., 1998
), and have been implicated in RNA splicing (Park et al., 2001
) and anti-apoptosis (Akahani et al., 1997
). A protein that binds both actin and mannose in vitro (Jung et al., 1996
) may be a cytoskeletal receptor for a cytoplasmic mannoprotein. Although the size of Skp1s glycan chain may evoke the expectation of a cognate receptor, complex cytoplasmic glycans might alternatively (1) regulate other modifications, such as phosphorylation as for O-ß-GlcNAc; (2) monitor protein folding as observed for N-glycans on proteins in the RER; or (3) reflect the availability of metabolic precursors required for the modification.
The characterization of cytoplasmic hydroxylation and glycosylation is technically challenging, but new MS-based proteomics approaches promise to overcome this barrier (Dell and Morris, 2001). As shown by the application of Q-TOF MS to both characterizing the glycan and sequencing rare GTases that modify Skp1, it is clear that complex cytoplasmic glycosylation has evolved in eukaryotic microorganisms and that this pathway is functionally important. There are strong hints of hydroxylation and complex glycosylation of other cytoplasmic/nuclear proteins in higher plants and animals and of cytoplasmic enzymes that might mediate these modifications. However, as previously observed for Golgi GTases, those of the cytoplasm seem to be equally evolutionarily divergent, necessitating a continuing need for new lead GTase sequences to seed BLAST searches and for refined informatics approaches to identify subtly related sequences already in the databases.
Acknowledgments
The authors are grateful to the many members of the glycobiology and Dictyostelium communities who have generously provided invaluable reagents and advice, including Anne Dell, Howard Morris, Monica Palcic, Jacques Baenziger, Adriana Manzi, Eric Holmes, Khushi Matta, Al Elbein, Bernard Henrissat, Gideon Davies, James van Etten, Hud Freeze, Bill Loomis, Dave Knecht, Rick Firtel, Frantisek Puta, and Gerry Weeks. The authors research in this area has been supported by grants from the NIH (GM-37539) and the Florida Division of the American Cancer Society.
Abbreviations
GTase, glycosyltransferase; MS, mass spectrometry; Q-TOF, hybrid quadrupole time-of-flight; PBCV-1, Paramecium bursaria Chlorella virus-1; PP, polypeptide; RER, rough endoplasmic reticulum; SCF; Skp1, cullin-1, F-box protein.
Footnotes
1 To whom correspondence should be addressed
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