Glycobiology Advance Access originally published online on July 31, 2008
Glycobiology 2008 18(11):861-870; doi:10.1093/glycob/cwn073
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Conservation of peptide acceptor preferences between Drosophila and mammalian polypeptide-GalNAc transferase ortholog pairs
2 Departments of Biochemistry and Pediatrics, Case Western Reserve University, Cleveland, OH 44106, USA
3 National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA
4 Department of Pediatrics, Case Western Reserve University, Cleveland, OH 44106, USA
1 To whom correspondence should be addressed: Tel: +1-216-368-4556; Fax: +1-216-368-4223; e-mail: txg2{at}cwru.edu
Received on May 23, 2008; revised on July 18, 2008; accepted on July 29, 2008
 |
Abstract |
|---|
 Top Abstract IntroductionResultsDiscussionMaterial and methodsSupplementary DataFundingConflict of interest statementReferences |
|---|
UDP-GalNAc:polypeptide
-N-acetylgalactosaminyltrans- ferases (ppGalNAc Ts) comprise a large family of glycosyltransferases that initiate mucin-type protein O-glycosylation, transferring -GalNAc to Thr and Ser residues of polypeptide acceptors. Families of ppGalNAc Ts are found across diverse eukaryotes with orthologs identifiable from mammals to single-cell organisms. The peptide substrate specificity and specific protein targets of the individual ppGalNAc T family members remain poorly understood. Previously, we reported a series of oriented random peptide substrate libraries for quantitatively determining the peptide substrate specificities of the mammalian ppGalNAc T1 and T2 (Gerken TA, Raman J, Fritz TA, Jamison O. 2006. Identification of common and unique peptide substrate preferences for the UDP-GalNAc:polypeptide -N-acetylgalactosaminyltransferases T1 & T2 (ppGalNAc T1 & T2) derived from oriented random peptide substrates. J Biol Chem. 281:32403–32416). With these substrates, previously unknown features of the transferases were revealed. Utilizing these and a new lengthened set of random peptides, studies have now been performed on PGANT5 and PGANT2, the Drosophila orthologs of T1 and T2. The results from these studies suggest that the major peptide substrate determinants for these transferases are contained within 2 to 3 residues flanking the site of glycosylation. It is further found that the mammalian and fly T1 orthologs display very similar peptide substrate preferences, while the T2 orthologs are nearly indistinguishable, suggesting similar peptide preferences amongst orthologous pairs have been maintained across evolution. This conclusion is further supported by sequence homology comparisons of each of the transferase orthologs, showing that the peptide substrate and UDP binding site residues are more highly conserved between species relative to their remaining catalytic and lectin domain residues. Key words: evolution / mucin / O-glycosylation / sequence motifs / UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase
|
Introduction |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterial and methodsSupplementary DataFundingConflict of interest statementReferences |
|---|
The first and defining step of mucin-type protein O-glycosylation, the transfer of
-GalNAc to the hydroxyl groups of Ser and Thr, is catalyzed by a large family of UDP-GalNAc:polypeptide -N-acetylgalactosaminyltransferases (ppGalNAc Ts). Members of the ppGalNAc T family appear in a wide range of organisms from single-cell parasites, worms, insects, and mammals. Most organisms possess multiple ppGalNAc T genes, with more than 20 members presently reported in mammals (Clausen and Bennett 1996
; Bennett et al. 1998, 1999; Ten Hagen et al. 1998, 1999, 2001; Toba et al. 2000; White et al. 2000; Guo et al. 2002; Schwientek et al. 2002; Ten Hagen and Tran 2002; Ten Hagen, Fritz, et al. 2003; Wang et al. 2003; Zhang et al. 2003; Cheng et al. 2004) and in the protein databases, 12 in Drosophila (Schwientek et al. 2002; Ten Hagen and Tran 2002; Ten Hagen, Tran, et al. 2003), and multiple members in C. elegans and other multicellular and single-cellular eukaryotes (Hagen and Nehrke 1998; Hagen et al. 2001; Wojczyk et al. 2003; Freire, Casaravilla, et al. 2003; Freire, Robello, et al. 2003; Stwora-Wojczyk, Dzierszinski, et al. 2004; Stwora-Wojczyk, Kissinger, et al. 2004; Freire et al. 2005). Peptide sequence comparisons between the mammalian and Drosophila families reveal four orthologous transferase pairs having between 67% and 86% sequence similarity in their conserved catalytic and carbohydrate-binding ricin-like domains (Schwientek et al. 2002; Ten Hagen and Tran 2002; Ten Hagen, Tran, et al. 2003). Such interspecies conservation suggests that these transferase orthologs may possess similar substrate preferences and may therefore play biologically significant roles that have remained conserved across evolution. Indeed, initial studies using a small panel of peptides and glycopeptide substrates have revealed what appears to be similar but not necessarily identical peptide and glycopeptide substrate specificities between the four conserved mammalian and fly transferases (Ten Hagen, Tran, et al. 2003). However, a detailed quantitative comparison of the substrate specificities of these tranferases demonstrating the extent of their similarities has not been performed. In this work, we report the first detailed comparison of the substrate specificities of two of the fly and mammalian orthologus pairs using a library of unmodified random peptide substrates.
Recently, we reported the use of a series of oriented random peptide substrates, GAGA(X)nT(X)nAGAGK (where X = randomized residue positions and n = 3, see Table I), for obtaining a nearly complete determination of the peptide substrate specificity of the mammalian ppGalNAc isoforms, T1 and T2 (Gerken et al. 2006). By comparing the relative compositions of the random residue positions in the lectin-isolated glycopeptide product with the nonglycosylated substrate, positional enrichment factors representative of the transferase preferences are obtained. These studies revealed common and unique position sensitive features for both transferases, several of which had not been previously detected. Using the transferase specific preferences, we were further able to generate unique optimum and selective acceptor peptide substrate sequences for each transferase. In the present work, we have utilized the above n = 3 random peptide substrates along with a new longer series of n = 5 substrates (see Table I) to characterize the substrate preferences of the Drosophila ppGalNAc T1 and T2 orthologs, PGANT5 and PGANT2, respectively. From these studies, the mammalian and fly T1 orthologs display very similar specificities while the T2 orthologs display nearly indistinguishable peptide substrate preferences. These findings suggest that the ppGalNAc T1 and particularly the ppGalNAc T2 orthologs may play related roles in both insects and mammals that have been maintained across evolution.
|
|
Results |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterial and methodsSupplementary DataFundingConflict of interest statementReferences |
|---|
ppGalNAc T1 and T2 enhancements for n = 5 random peptides VI and VII
ppGalNAc T1 and T2 enhancement values were obtained against peptides VI and VII as described for peptides I, III, IV, and V (Gerken et al. 2006
). The composition of these peptides were chosen to cover the same 16-amino-acid residues characterized by the n = 3 peptides I, III, IV, and V, but by using only two different peptides. As shown in supplemental Figure S1, the enhancements obtained for the n = 5 peptides, VI and VII, are in good agreement with the enhancements previously obtained from the shorter, n = 3, random peptides for both ppGalNAc T1 and T2 (correlation coefficients, r2, of 0.80 and 0.89 for ppGalNAc T1 and T2, respectively, Table II). These results show that the random peptide transferase preferences for the flanking + or –3 residues of the site of glycosylation are not altered with increased peptide substrate length nor are they significantly influenced by the proximity of the flanking alternating Gly and Ala residues. Therefore, the n = 5 and n = 3 enhancement values for all six random peptides listed in Table I were combined and their average values plotted in Figures 1 and 2 for both ppGalNAc T1 and T2 (see panels A and C of each figure for the hydrophobic and hydrophylic residues, respectively). The plots further suggest that ppGalNAc T1 and T2 are for the most part relatively insensitive (i.e., enhancement values predominantly 
1) to the nature of the amino acid residues at positions remote from the site of glycosylation (i.e., 4 to 5 residues away).
|
|
|
Drosophila transferase PGANT5 and PGANT2 enhancements for the n = 3 and n = 5 random peptides PI and PIII – PVII
The four n = 3 random peptide substrates previously utilized for characterizing the specificity of ppGalNAC T1 and T2 (Gerken et al. 2006
), along with the two n = 5 random peptides (Table I) were glycosylated by media collected from COS7 cells expressing PGANT5 and PGANT2 (see Materials and methods section) and their enhancements obtained (Gerken et al. 2006). To demonstrate that the observed preferences do not reflect the presence of contaminating transferases from the COS7 cells, two additional experiments were performed. In the first experiment, media from COS7 cells mock transfected with empty vector were shown to give no significant radio-labeled random glycopeptide product on Sephadex G10 chromatography (data not shown). In the second, FLAG-tagged fly transferases isolated and purified on an anti-FLAG affinity gel were shown to give identical enhancement patterns as those obtained from media alone (1–2 determinations for each transferase for peptides VI and VII). We therefore conclude that the obtained preferences accurately represent the preferences of the authentic fly transferases. The obtained preferences for PGANT5 and PGANT2 averaged for all six peptides are plotted in Figures 1 and 2 (see panels B and D of each figure for the hydrophobic and hydrophylic residues, respectively).
Comparison of the mammalian and Drosophila transferase T1 and T2 orthologs (Figures 1 and 2, and Supplemental Figures S2–S5)
As shown in Figure 1, the Drosophila PGANT5 displays very similar enhancement patterns relative to the mammalian ppGalNAc T1. Generally, the ranking of hydrophobic residue enhancements at each position remains the same for both transferases, although the magnitude of the enhancements values may vary between transferases (see supplemental Figure S2 for individual hydrophobic residue comparisons). For example, large Pro enhancements are observed at the –1, +1, and +3 positions, moderate Val and Tyr enhancements are found at the –1 and +3 positions, respectively, and Met is uniquely disfavored at the –1 site for both transferases. The hydrophylic residues on the other hand show more variability between transferases, although both transferases show low hydrophylic preferences at the –1 position and moderate to strong Glu/Asp preferences at the +1 position (see supplemental Figure S3). The largest difference between tranferases is the nearly 2-fold Glu enhancement in PGANT5 at the +1 and +2 positions compared to ppGalNAc T1. A plot of the PGANT5 versus ppGalNAc T1 enhancements shown in Figure 3A gives a correlation coefficient, r2, of 0.80 (Table II). The plots in Figure 3B and C further demonstrate the differences in the correlation of the hydrophobic and hydrophylic residue enhancements giving r2 values of 0.86 and 0.64, respectively (Table II).
|
A comparison of the ppGalNAc T2 and PGANT2 preferences (Figures 2 and Figure 3D–F and supplemental Figures S4 and S5) reveals nearly indistinguishable preferences. For example, the plot of the fly versus mammalian T2 transferases, Figure 3D, gives a correlation coefficient r2 of 0.92 for all residues. (Table II) Hydrophobic residues display an even higher correlation (r2 = 0.95) whereas the hydrophylic residues display a somewhat poorer correlation (r2 = 0.69) presumably due to the difficulties in quantifying Lys, Arg, and His residues. (Figure 3E and F, Table II). It is worth noting that the correlations between the mammalian and fly T2 transferases are generally equal to or better than what was found between the n = 3 and n = 5 random peptide substrates glycosylated by ppGalNAc T2 (Table II). For all practical purposes, we conclude that the fly PGANT2 and human ppGalNAc T2 possess the same peptide substrate preferences that have not deviated significantly over evolution, while the fly PGANT5 and mammalian ppGalNAc T1 preferences that have changed slightly.
Comparison of the fly and mammalian ppGalNAc T peptide sequences
Having two pairs of transferases with known peptide substrate specificites now allows us to examine the relationship between ppGalNAc T sequence and peptide substrate specificity. In our previous work (Table III and Figure S8 (Gerken et al. 2006)), we compared 17 residues in ppGalNAc T1 and T2 that were estimated to be in direct contact (within 5Å) of the bound peptide substrate based on the crystal structure of human ppGalNAc T2 bound to the EA2 peptide (Fritz et al. 2006). To further this analysis, we have performed sequence alignments on the catalytic and lectin domains of PGANT5 and PGANT2 against their human ppGalNAc T orthologs. In the present work, we examined the residue conservation in arbitrary zones of increasing distance from the peptide binding site. These zones (listed in Table III) encompassed: 20 direct substrate interacting residues (P20); 97 residues underlying the peptide binding site (P97); 196 residues within 20Å of Thr7 of the bound EA2 peptide (P196); and the entire catalytic domain of 358 residues (Cat). In addition, the 127 lectin domain residues (Lect), the 495 catalytic to lectin domain residues (C+L), and the 13 direct (Fritz et al. 2006), and 42 underlying UDP binding residues (U13 and U42, respectively) were also assessed for their conservation. In Table III, the percent residue identity for the P20 and P97 zones were also broken down into N10, C10, N 48, and C49 regions representing residues interacting with the N- and C-terminal portions of the peptide substrate relative to the GalNAc acceptor site (i.e., residue Thr 7, of the bound EA2 peptide (Fritz et al. 2006)). These residues and regions are highlighted on the structure of ppGalNAc T2 in supplemental Figure S6. The sequence alignments from which the P20, P97, P196, U13, and U42 homologies were derived are given in supplemental Table SI. Similar to Table III, a list of residue similarity and nonconservation for each zone is given in supplemental Table SII. Because of the very high identity (87%) of the human ppGalNAc T1 to ppGalNAc T13, we have also included this comparison in the tables.
|
The goals of the analysis (summarized in Table III and supplemental Table SII) were to compare the extent of residue conservation surrounding the peptide binding/catalytic site versus regions more remote from the peptide binding cleft. It was anticipated that this analysis would provide a sense of the extent that transferase pairs were evolutionarily constrained to maintain their peptide substrate specificity and peptide binding site. Remarkably, the ppGalNAc T2 and PGANT2 pair shows 80 to 90% residue identity in the 20 directly binding residues (P20) through to the 196 surrounding residues 20Å from the substrate acceptor site (P196) (Table III). At the level of the full
358 residue catalytic domain (Cat), the percent residue identity decreases to 73% for the T2 pair, suggesting that residues more remote from the peptide binding/catalytic site have undergone more evolutionary drift than the residues comprising the peptide binding site. Similarly, the lectin domain (Lect) shows even greater evolutionary drift having only 47% of its residues conserved. For additional comparison, both the direct and underlying UDP binding residues (U13 and U42) are highly conserved (100 and 88%, respectively) for the T2 pair (Table III). The very high conservation of the peptide and UDP binding residues in the T2 transferase pair is entirely consistent with their nearly indistinguishable random peptide substrate preferences (Figure 2 and Table II).
By comparison, the ppGalNAc T1 and PGANT5 pair displays significantly lower sequence identities, 50–70%, at the level of the 20 directly binding residues (P20), although identity increases for the 97 underlying residues (P97) and reaches 81% at the 196 residue 20Å level (P196) (see Table III). The relatively high identity for the latter is unlikely due to a general conservation of the overall sequence of the peptide binding region in all the ppGalNAc Ts, as the identity between the T1 and T2 ortholog pairs for this P97 and P196 regions is significantly lower (60%) (Table III). Similar to the T2 pair, the full catalytic domain and lectin domains of the T1 ortholog pair show decreased identities compared to the largest (P196) peptide binding region, i.e., 71 and 54% identity, respectively, compared to 81%. Again the direct and underlying UDP binding residues are highly conserved between ppGalNAc T1 and PGANT5 with 92–95% identity, respectively.
|
Discussion |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterial and methodsSupplementary DataFundingConflict of interest statementReferences |
|---|
Using a series of oriented random peptide substrate acceptors (Table I), we have characterized the detailed peptide substrate specificities of two transferases from the fly, PGANT5 and PGANT2, whose mammalian orthologs, ppGalNAc T1 and T2, have been previously characterized (Gerken et al. 2006
). Additionally, we have introduced a series of longer, n = 5, random peptide substrates thereby extending the characterization of each isoform to plus and minus five residues from the site of glycosylation. The longer n = 5 peptides give identical preferences as those previously determined with the n = 3 peptides (Gerken et al. 2006), thus validating their use and further demonstrating that the flanking Gly-Ala repeats do not alter the transferase specificity in these substrates. Our results further show that the major peptide substrate determinants lie within the 2 to 3 residues flanking the site of glycosylation for the T1 and T2 isoforms. These findings are consistent with recent molecular dynamics studies of ppGalNAc T2 bound to the EA2 substrate which suggests that residues at the +4 to +6 positions of the substrate peptide are more flexible and less tightly bound than the peptide substrate residues surrounding the site of glycosylation (Milac et al. 2007). In summary, we now have a series of random peptide substrates suitable for the quantitative characterization of the peptide substrate specificity of individual ppGalNAc Ts. As shown in Figures 1–3 and supplemental Figures S2–S5 the peptide substrate preferences obtained for the fly PGANT5 and PGANT2 were very similar to their mammalian orthologs, ppGalNAc T1 and T2, which show 71% and 73% sequence identity in their catalytic domains, respectively (Table III). Indeed, the differences between the fly PGANT2 and human ppGalNAc T2 are indistinguishable within an experimental error. For the PGANT5 and ppGalNAc T1 transferase pair, the most significant differences are elevated Glu enhancements at the +1 and +2 positions. This suggests that evolutionary pressures have maintained relatively tightly, the peptide substrate specificities of these transferases from insects to mammals. In further support of the biological importance of the conservation of these transferases, ppGalNAc T1, T2, and T7 are shown to have some of the lowest rates of nonsynonymous nucleotide substitutions between human and rodents among 55 glycosyltransferases that were studied (Kaneko et al. 2001). Significantly, these three transferases also have close orthologs in the fly (Ten Hagen, Tran, et al. 2003).
It is well established that a number of ppGalNAc transferases, i.e., T1, T2, and T4, possess variably altered preferences against certain monosaccharide GalNAc containing mucin or glycopeptide substrates when compared to their nonglycosylated analogs and that these differences are due to the presence of an intact lectin domain (Hagen et al. 1999; Hassan et al. 2000; Tenno, Kezdy, et al. 2002; Tenno, Saeki, et al. 2002; Fritz et al. 2006; Wandall et al. 2007; Raman et al. 2008). Truncation and mutagenesis studies have further demonstrated that the lectin binding domain is not required for ppGalNAc T1 or T2 catalysis or peptide substrate specificity (Hagen et al. 1999; Fritz et al. 2006; Raman et al. 2008). The inhibition of ppGalNAc T1 and T2 by neighboring glycosylation (Gerken et al. 2002, 2004) and the glycopeptide substrate profiling studies of Pratt et al. (2004), suggesting that T1 and T2 are early or initiating transferases, are consistent with the idea that the biological substrates for these transferases indeed may not require prior glycosylation. Our comparison of the human and fly ppGalNAc T1 and T2 orthologs reveals that the lectin domains of either transferase pair are considerably less conserved compared to their catalytic domain (Table III). Therefore, it seems that for these transferases the conservation of the function of their lectin domains may not have been as critical as the conservation of the peptide substrate specificities of their catalytic domains. Indeed, our earlier studies show that ppGalNAc T1 can glycosylate glycopeptide acceptors that PGANT5 cannot, while ppGalNAc T2 and PGANT2 appear to have similar but not identical glycopeptide preferences (Ten Hagen, Tran, et al. 2003). Thus, the fly and mammalian ppGalNAc T1 and T2 orthologs have largely maintained their respective peptide substrate specificities while seemingly diverging in their lectin domain-mediated glycopeptide specificities.
One might expect that the most likely targets for these transferases would be the initiation of the glycosylation of mucins and mucin-like molecules containing heavily O-glycosylated mucin tandem repeat domains, commonly called PTS domains for their high Pro, Thr, and Ser content. However, the peptide sequences of the PTS domains, even for the same mucins, have rapidly evolved within related mammalian or insect species (Lang et al. 2007; Carmon et al. 2007). Both the mammalian and fly mucin PTS domains commonly possess a range of acceptable motifs for both ppGalNAc T1 and T2; however, the number of these motifs varies considerably among mucins. In addition, ppGalNAc T1 and T2 are expressed across a range of mucin and nonmucin secreting tissues in the mouse (Young et al. 2003) and across tissues in the fly (Tian and Ten Hagen 2006). Hence, it is difficult to envision how such a high degree of sequence variability could drive the relatively tight conservation of the peptide substrate specificities of these transferases. However, there are several recent examples where protein glycosylation by specific ppGalNAc Ts may be required for biological function (Topaz et al. 2004; Kato et al. 2006; Wagner et al. 2007; Herr et al. 2008). Therefore, it would not be surprising for ppGalNAc T1 and T2, and their insect orthologs, to possess one or more unique protein targets to glycosylate, in addition to contributing to the initiation of mucin PTS domain glycosylation. Toward the very difficult goal of identifying potential protein targets, collaborative proteomics studies on transferase knockouts in the fly and mouse are underway. Work is also in progress developing a protein sequence database searching strategy for searching and scoring potential sites of transferase specific glycosylation to complement these studies.
|
Material and methods |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterial and methodsSupplementary DataFundingConflict of interest statementReferences |
|---|
Random peptide substrates, GAGA(X)nT(X)nAGAGK, where n = 3 or 5 and X the randomized residues given in Table I, were custom synthesized by QCB Inc. Hopkinton, MA. The rational for the design of these peptides has been previously discussed (Gerken et al. 2006
). Purified recombinant mammalian ppGalNAc T1 (bovine) (Elhammer et al. 1999; Homa et al. 1995) and T2 (human) (Fritz et al. 2006) were kind gifts of Drs. Ake Elhammer (Kalamazoo, MI) and Lawrence Tabak (Office of Director, NIDCR, National Institutes of Health, Bethesda, MD), respectively. Their purity and kinetic properties have been described previously (Gerken et al. 2006). (Note that in this work the bovine ppGalNAc T1 has been utilized. Its amino acid sequence is 99% identical to the human transferase, with no residue differences in the peptide binding site regions described in Table III.) Mammalian ppGalNAc T incubations with the n = 5 random peptide substrates were performed as previously described for these transferases for the n = 3 random peptide substrates (Gerken et al. 2006). Drosophila melanogaster transferases were expressed in COS-7 cells after LipofectAMINE (Invitrogen) transfection with the pIMKF4 SV40 secretion expression vector containing an insulin secretion signal sequence, metal-binding and kinase sites, and a FLAG epitope site followed by the transferase of interest lacking its N-terminal transmembrane domain as described (Hagen et al. 1997; Ten Hagen, Tran, et al. 2003). Media (48–72 h posttransfection) containing secreted transferase were collected and frozen at –80°C in small aliquots to minimize free-thaw cycles. Drosophila transferase reactions (100 µL) typically contained 10 mg/mL random peptide (n = 3 or 5), 2 mM UDP-GalNAc (3H-labeled), 10 mM MnCl2, 60 µL COS-7 media containing transferase, 100 mM HEPES, pH 7.5, and protease inhibitors (Sigma #P8340 and P8849) diluted by 1/12 of their original concentration. Overnight incubations at 37°C were quenched with EDTA and passed through Dowex 1-X8 to remove unreacted UDP-GalNAc as previously described (Gerken et al. 2006). Fly transferase reactions were also performed on the n = 5 random peptides (Table I) using transferase bound to anti-FLAG M2 agarose affinity gel (Sigma A2220). Briefly 100 µL of settled anti-FLAG gel (previously washed with 0.12 M glycine, pH 3.5, followed by 50 mM Tris, pH 7.4, 150 mM NaCl following the manufacturer's recommendations) was rocked for 3 h in the cold with 0.5 mL of COS7 cell media (made 150 mM NaCl) containing the FLAG-labeled fly transferase. After exhaustive washing of the gel with a Tris buffer, 40 µL of settled gel was transferred to a 0.5 mL Eppendorf tube to which 60 µL of the standard transferase reaction mixture containing random peptide, UDP-GalNAc, and buffers (as described above) were added. The tubes were shaken overnight at 37°C in a shaking microplate incubator (Taitec Microincubator M-36, Taitec Instruments, San Jose, CA) at a setting suitable for maintaining the gel beads in suspension. Reactions were stopped with EDTA and passed through Dowex 1-X8 as described above.
Typically the extent of random peptide glycosylation was 3% or less based on 3H-GalNAc incorporation for both the media and affinity gel bound transferase reactions. Control vector media reactions revealed insignificant incorporation of 3H-GalNAc into the random peptide substrates compared to transferase containing media. Subsequent Sephadex G10 and mixed bed lectin affinity chromatographic procedures isolating the glycosylated random peptide were performed as previously described (Gerken et al. 2006). Random peptides and glycopeptides were sequenced on a Procise 494 Edman amino acid sequencer as previously described (Gerken et al. 2006); however, the n = 5 random peptides Pro sequence cycles were employed at each of X positions to help reduce sequence lag. Amino acid residue enhancement factors were obtained at each randomized residue position by comparing the X residue glycopeptide mole fractions to the peptide mole fractions obtained prior to the lectin column chromatography (Gerken et al. 2006). As discussed previously (Gerken et al. 2006), the Trp residues in peptide V (Table I) are lost due to oxidation; therefore, their enrichment values were not obtained.
Transferase sequence multiple alignments and identification of catalytic and lectin domains based on X-ray crystal structures of T1 and T2
Blast peptide sequence searches using human ppGalNAc T1 and T2 sequences as templates were performed using the NCBI (National Center for Biothechnology Information) blastp protein–protein blast search tool (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) using the nonredundant protein sequences database. Sequences were also aligned using web-based utilities CLUSTALW and MULTALIN at the The PBIL (Pôle Bio-Informatique Lyonnais) World Wide Web server: http://pbil.univ-lyon1.fr/ and by Tcoffee at the CNRS (Centre National de la Recherche Scientifique) at http://www.igs.cnrs-mrs.fr/Tcoffee/tcoffee_cgi/index.cgi the latter using the ppGalNAc T2 bound to EA2 structure as template (Fritz et al. 2006). Identification of the catalytic and lectin domain boundaries as well as the UDP and peptide binding residues were taken from the published analysis of the X-ray structures of ppGalNAc T1 and T2 (Fritz et al. 2004, 2006). Sequence pattern searches were also performed using FUZZPRO (BioTeam, iNquiry Bioinformatics Portal) on a nonredundant database of all secreted fly and human protein sequences.
|
Supplementary Data |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterial and methodsSupplementary DataFundingConflict of interest statementReferences |
|---|
Supplementary data for this article is available online at http://glycob.oxfordjournals.org/.
|
Funding |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterial and methodsSupplementary DataFundingConflict of interest statementReferences |
|---|
National Institutes of Health (Grant RO1-CA-78834 to T.A.G.); the Intramural Research Program of the National Institute of Dental and Craniofacial Research, National Institutes of Health (to K.G.T.H).
|
Conflict of interest statement |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterial and methodsSupplementary DataFundingConflict of interest statementReferences |
|---|
None declared.
|
Acknowledgements |
|---|
We acknowledge the generous gift of mammalian transferases by Drs. Ake Elhammer (Kalamazoo, MI) and Lawarence Tabak, Timothy Fritz, and Jayalakshmi Raman (National Institute of Dental and Craniofacial Research and National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD). We also acknowledge Dr. Mark Adams (Department of Genetics, Case Western Reserve University) for constructing fly and human secreted protein databases and Chin Park for contributing to the initial studies.
|
Abbreviations |
|---|
ppGalNAc T;, ; mammalian UDP-GalNAc:polypeptide
-N-acetylgalactosaminyltransferases, PGANT; Drosphila UDP-GalNAc:polypeptide -N-acetylgalactosaminyltransferases, |
References |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterial and methodsSupplementary DataFundingConflict of interest statementReferences |
|---|
Bennett EP, Hassan H, Mandel U, Hollingsworth MA, Akisawa N, Ikematsu Y, Merkx G, van Kessel AG, Olofsson S, Clausen H. Cloning and characterization of a close homologue of human UDP-N-acetyl-
-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-T3, designated GalNAc-T6. Evidence for genetic but not functional redundancy. J Biol Chem (1999) 274:25362–25370.Bennett EP, Hassan H, Mandel U, Mirgorodskaya E, Roepstorff P, Burchell J, Taylor-Papadimitriou J, Hollingsworth MA, Merkx G, van Kessel AG, et al. Cloning of a human UDP-N-acetyl--D-galactosamine:polypeptide N-acetylgalactosaminyltransferase that complements other GalNAc- transferases in complete O-glycosylation of the MUC1 tandem repeat. J Biol Chem (1998) 273:30472–30481.
Carmon A, Wilkin M, Hassan J, Baron M, MacIntyre R. Concerted evolution within the Drosophila dumpy gene. Genetics (2007) 176:309–325.
Cheng L, Tachibana K, Iwasaki H, Kameyama A, Zhang Y, Kubota T, Hiruma T, Tachibana K, Kudo T, Guo JM, et al. Characterization of a novel human UDP-GalNAc transferase, pp-GalNAc-T15. FEBS Lett (2004) 566:17–24.[CrossRef][Web of Science][Medline]
Clausen H, Bennett EP. A family of UDP-GalNAc:polypeptide N-acetylgalactosaminyl-transferases control the initiation of mucin-type O-linked glycosylation. Glycobiology (1996) 6:635–646.
Elhammer AP, Kezdy FJ, Kurosaka A. The acceptor specificity of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases. Glycoconj J (1999) 16:171–180.[CrossRef][Web of Science][Medline]
Freire T, Casaravilla C, Carmona C, Osinaga E. Mucin-type O-glycosylation in Fasciola hepatica: Characterisation of carcinoma-associated Tn and sialyl-Tn antigens and evaluation of UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase activity. Int J Parasitol (2003) 33:47–56.[CrossRef][Web of Science][Medline]
Freire T, Fernandez C, Chalar C, Maizels RM, Alzari P, Osinaga E, Robello C. Characterization of a UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase with an unusual lectin domain from the platyhelminth parasite Echinococcus granulosus. Biochem J (2005) 382:501–510.[CrossRef][Web of Science]
Freire T, Robello C, Soule S, Ferreira F, Osinaga E. Sialyl-Tn antigen expression and O-linked GalNAc-Thr synthesis by Trypanosoma cruzi. Biochem Biophys Res Commun (2003) 312:1309–1316.[CrossRef][Web of Science][Medline]
Fritz TA, Hurley JH, Trinh LB, Shiloach J, Tabak LA. The beginnings of mucin biosynthesis: The crystal structure of UDP-GalNAc:polypeptide -N-acetylgalactosaminyltransferase-T1. Proc Natl Acad Sci USA (2004) 101:15307–15312.
Fritz TA, Raman J, Tabak LA. Dynamic association between the catalytic and lectin domains of human UDP-GalNAc:polypeptide -N-acetylgalactosaminyltransferase-2. J Biol Chem (2006) 281:8613–8619.
Gerken TA, Raman J, Fritz TA, Jamison O. Identification of common and unique peptide substrate preferences for the UDP-GalNAc:polypeptide -N-acetylgalactosaminyltransferases T1 & T2 (ppGalNAc T1 & T2) derived from oriented random peptide substrates. J Biol Chem (2006) 281:32403–32416.
Gerken TA, Tep C, Rarick J. Role of peptide sequence and neighboring residue glycosylation on the substrate specificity of the uridine 5'-diphosphate--N-acetylgalactosamine:polypeptide N-acetylgalactosaminyl transferases T1 and T2: Kinetic modeling of the porcine and Canine Submaxillary Gland mucin tandem repeats. Biochemistry (2004) 43:9888–9900.[CrossRef][Web of Science][Medline]
Gerken TA, Zhang J, Levine J, Elhammer A. Mucin core O-glycosylation is modulated by neighboring residue glycosylation status: Kinetic modeling of the site-specific glycosylation of the apo-porcine sumbaxillary mucin tandem repeat by UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases T1 and T2. J Biol Chem (2002) 277:49850–49862.
Guo JM, Zhang Y, Cheng L, Iwasaki H, Wang H, Kubota T, Tachibana K, Narimatsu H. Molecular cloning and characterization of a novel member of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family, pp-GalNAc-T12. FEBS Lett (2002) 524:211–218.[CrossRef][Web of Science][Medline]
Hagen FK, Hazes B, Raffo R, deSa D, Tabak LA. Structure-function analysis of the UDP-N-acetyl-D- galactosamine:polypeptide N-acetylgalactosaminyltransferase. Essential residues lie in a predicted active site cleft resembling a lactose repressor fold. J Biol Chem (1999) 274:6797–6803.
Hagen FK, Layden M, Nehrke K, Gentile K, Berbach K, Tsao CC, Forsythe M. Mucin-type O-glycosylation in C. elegans is initiated by a family of glycosyltransferases. Trends Cell Biol (2001) 13:463–479.[CrossRef]
Hagen FK, Nehrke K. cDNA Cloning and expression of a family of UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase sequence homologs from Caenorhabditis elegans. J Biol Chem (1998) 273:8268–8277.
Hagen FK, Ten Hagen KG, Beres TM, Balys MM, VanWuyckhuyse BC, Tabak LA. cDNA cloning and expression of a novel UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase. J Biol Chem (1997) 272:13843–13848.
Hassan H, Reis CA, Bennett EP, Mirgorodskaya E, Roepstorff P, Hollingsworth MA, Burchell J, Taylor-Papadimitriou J, Clausen H. The lectin domain of UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-T4 directs its glycopeptide specificities. J Biol Chem (2000) 275:38197–38205.
Herr P, Korniychuk G, Yamamoto Y, Grubisic K, Oelgeschlager M. Regulation of TGF-β signalling by N-acetylgalactosaminyltransferase-like 1. Development (2008) 135:1813–1822.
Homa FL, Baker CA, Thomsen DR, Elhammer AP. Conversion of a bovine UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase to a soluble, secreted enzyme, and expression in Sf9 cells. Protein Expr Purif (1995) 6:141–148.[CrossRef][Web of Science][Medline]
Kaneko M, Nishihara S, Narimatsu H, Saitou N. The evolutionary history of glycosyltransferase genes. Trends Glycosci Glycotechnol (2001) 13:147–155.
Kato K, Jeanneau C, Tarp MA, Benet-Pages A, Lorenz-Depiereux B, Bennett EP, Mandel U, Strom TM, Clausen H. Polypeptide GaINAc-transferase T3 and familial tumoral calcinosis: Secretion of FGF23 requires O-glycosylation. J Biol Chem (2006) 281:18370–18377.
Lang T, Hansson GC, Samuelsson T. Gel-forming mucins appeared early in metazoan evolution. Proc Natl Aacd Sci (2007) 104:16209–16214.[CrossRef]
Milac AL, Buchete NV, Fritz TA, Hummer G, Tabak LA. Substrate-induced conformational changes and dynamics of UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase-2. J Mol Biol (2007) 373:439–451.[CrossRef][Web of Science][Medline]
Pratt MR, Hang HC, Ten Hagen KG, Rarick J, Gerken TA, Tabak LA, Bertozzi CR. Deconvoluting the functions of polypeptide N--acetylgalactosaminyltransferase family members by glycopeptide substrate profiling. Chem Biol (2004) 11:1009–1016.[CrossRef][Web of Science][Medline]
Raman J, Fritz TA, Gerken TA, Jamison O, Live D, Lu M, Tabak LA. The catalytic and lectin domains of UDP-GalNAc:polypeptide -N-acetylgalactosaminyltransferase function in concert to direct glycosylation site selection. J Biol Chem (2008) 283:22942–22951.
Schwientek TJ, Bennett EP, Flores C, Thacker J, Hollman M, Reis CA, Behrens J, Mandel U, Keck B, Schafer MA, et al. Functional conservation of subfamilies of putative UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases in Drosophila, C. elegans and mammals: One subfamily comprised of l(2)35Aa is essential in Drosophila. J Biol Chem (2002) 277:22623–22638.
Stwora-Wojczyk MM, Dzierszinski F, Roos DS, Spitalnik SL, Wojczyk BS. Functional characterization of a novel Toxoplasma gondii glycosyltransferase: UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-T3. Arch Biochem Biophys (2004) 426:231–240.[CrossRef][Web of Science][Medline]
Stwora-Wojczyk MM, Kissinger JC, Spitalnik SL, Wojczyk BS. O-Glycosylation in Toxoplasma gondii: Identification and analysis of a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases. Int J Parasitol (2004) 34:309–322.[CrossRef][Web of Science][Medline]
Ten Hagen KG, Bedi GS, Tetaert D, Kingsley PD, Hagen FK, Balys MM, Beres TM, Degand P, Tabak LA. Cloning and characterization of a ninth member of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family, ppGaNTase-T9. J Biol Chem (2001) 276:17395–17404.
Ten Hagen KG, Fritz TA, Tabak LA. "All in the family"– The UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases. Glycobiology (2003) 13:1R–16R.
Ten Hagen KG, Hagen FK, Balys MM, Beres TM, Van Wuyckhuyse B, Tabak LA. Cloning and expression of a novel, tissue specifically expressed member of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family. J Biol Chem (1998) 273:27749–27754.
Ten Hagen KG, Tetaert D, Hagen FK, Richet C, Beres TM, Gagnon J, Balys MM, VanWuyckhuyse B, Bedi GS, Degand P, et al. Characterization of a UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase that displays glycopeptide N-acetylgalactosaminyltransferase activity. J Biol Chem (1999) 274:27867–27874.
Ten Hagen KG, Tran DT. A UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase is essential for viability in Drosophila melanogaster. J Biol Chem (2002) 277:22616–22622.
Ten Hagen KG, Tran DT, Gerken TA, Stein DS, Zhang Z. Functional characterization and expression analysis of members of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family from Drosophila melanogaster. J Biol Chem (2003) 278:35039–35048.
Tenno M, Kezdy FJ, Elhammer AP, Kurosaka A. Function of the lectin domain of polypeptide N-acetylgalactosaminyltransferase 1. Biochem Biophys Res Commun (2002) 298:755–759.[CrossRef][Web of Science][Medline]
Tenno M, Saeki A, Kezdy FJ, Elhammer AP, Kurosaka A. The lectin domain of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 1 is involved in O-glycosylation of a polypeptide with multiple acceptor sites. J Biol Chem (2002) 277:47088–47096.
Tian E, Ten Hagen KG. Expression of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family is spatially and temporally regulated during Drosophila development. Glycobiology (2006) 16:83–95.
Toba S, Tenno M, Konishi M, Mikami T, Itoh N, Kurosaka A. Brain-specific expression of a novel human UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAc-T9). Biochimica et Biophysica Acta (2000) 1493:264–268.[Medline]
Topaz O, Shurman DL, Bergman R, Indelman M, Ratajczak P, Mizrachi M, Khamaysi Z, Behar D, Petronius D, Friedman V, et al. Mutations in GALNT3, encoding a protein involved in O-linked glycosylation, cause familial tumoral calcinosis. Nat Genet (2004) 36:579–581.[CrossRef][Web of Science][Medline]
Wagner KW, Punnoose EA, Januario T, Lawrence DA, Pitti RM, Lancaster K, Lee D, von Goetz M, Yee SF, Totpal K, et al. Death-receptor O-glycosylation controls tumor-cell sensitivity to the proapoptotic ligand Apo2L/TRAIL. Nat Med (2007) 13:1070–1077.[CrossRef][Web of Science][Medline]
Wandall HH, Irazoqui F, Tarp MA, Bennett EP, Mandel U, Takeuchi H, Kato K, Irimura T, Suryanarayanan G, Hollingsworth MA, et al. The lectin domains of polypeptide GalNAc-transferases exhibit carbohydrate-binding specificity for GalNAc: Lectin binding to GalNAc-glycopeptide substrates is required for high density GalNAc-O-glycosylation. Glycobiology (2007) 17:374–387.
Wang H, Tachibana K, Zhang Y, Iwasaki H, Kameyama A, Cheng L, Guo J, Hiruma T, Togayachi A, Kudo T, et al. Cloning and characterization of a novel UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, pp-GalNAc-T14. Biochem Biophys Res Commun (2003) 300:738–744.[CrossRef][Web of Science][Medline]
White KE, Lorenz B, Evans WE, Meitinger T, Strom TM, Econs MJ. Molecular cloning of a novel human UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, GalNAc-T8, and analysis as a candidate autosomal dominant hypophosphatemic rickets (ADHR) gene. Gene (2000) 246:347–356.[CrossRef][Web of Science][Medline]
Wojczyk BS, Stwora-Wojczyk MM, Hagen FK, Striepen B, Hang HC, Bertozzi CR, Roos DS, Spitalnik SL. cDNA cloning and expression of UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase T1 from Toxoplasma gondii. Mol Biochem Parasitol (2003) 131:93–107.[CrossRef][Web of Science][Medline]
Young WW Jr, Holcomb DR, Ten Hagen KG, Tabak LA. Expression of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase isoforms in murine tissues determined by real-time PCR: A new view of a large family. Glycobiology (2003) 13:549–557.
Zhang Y, Iwasaki H, Wang H, Kudo T, Kalka TB, Hennet T, Kubota T, Cheng L, Inaba N, Gotoh M, et al. Cloning and characterization of a new human UDP-N-acetyl--D-galactosamine:polypeptide N-acetylgalactosaminyltransferase, designated pp-GalNAc-T13, that is specifically expressed in neurons and synthesizes Tn antigen. J Biol Chem (2003) 278:573–584.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  C. L. Perrine, A. Ganguli, P. Wu, C. R. Bertozzi, T. A. Fritz, J. Raman, L. A. Tabak, and T. A. Gerken Glycopeptide-preferring Polypeptide GalNAc Transferase 10 (ppGalNAc T10), Involved in Mucin-type O-Glycosylation, Has a Unique GalNAc-O-Ser/Thr-binding Site in Its Catalytic Domain Not Found in ppGalNAc T1 or T2 J. Biol. Chem., July 24, 2009; 284(30): 20387 - 20397. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Perrine, T. Ju, R. D Cummings, and T. A Gerken Systematic determination of the peptide acceptor preferences for the human UDP-Gal:glycoprotein-{alpha}-GalNAc {beta}3 galactosyltranferase (T-synthase) Glycobiology, March 1, 2009; 19(3): 321 - 328. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. G. T. Hagen, L. Zhang, E Tian, and Y. Zhang Glycobiology on the fly: Developmental and mechanistic insights from Drosophila Glycobiology, February 1, 2009; 19(2): 102 - 111. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||