Posttranslational modifications of bovine osteopontin: identification of twenty-eight phosphorylation and three O-glycosylation sites.

Osteopontin (OPN) is a multiphosphorylated glycoprotein found in bone and other normal and malignant tissues, as well as in the physiological fluids urine and milk. The present study demonstrates that bovine milk osteopontin is phosphorylated at 27 serine residues and 1 threonine residue. Phosphoamino acids were identified by a combination of amino acid analysis, sequence analysis of S-ethylcysteine-derivatized phosphopeptides, and mass spectrometric analysis. Twenty-five phosphoserines and one phosphothreonine were located in Ser/Thr-X-Glu/Ser(P)/Asp motifs, and two phosphoserines were found in the sequence Ser-X-X-Glu/Ser(P). These sequence motifs are identical with the recognition sequences of mammary gland casein kinase and casein kinase II, respectively. Examination of the phosphorylation pattern revealed that the phosphorylations were clustered in groups of approximately three spanned by unphosphorylated regions of 11-32 amino acids. This pattern is probably of importance in the multiple functions of OPN involving interaction with Ca2+ and inorganic calcium salts. Furthermore, three O-glycosylated threonines (Thr 115, Thr 124, and Thr 129) have been identified in a threonine- and proline-rich region of the protein. Three putative N-glycosylation sites (Asn 63, Asn 85, and Asn 193) are present in bovine osteopontin, but sequence and mass spectrometric analysis showed that none of these asparagines were glycosylated in bovine mammary gland osteopontin. Alignment analysis showed that the majority of the phosphorylation sites in bovine osteopontin as well as all three O-glycosylation sites were conserved in other mammalian sequences. This conservation of serines, even in otherwise less well-conserved regions of the protein, indicates that the phosphorylation of osteopontin at specific sites is essential for the function of the protein.     

Emerging paradigms for the initiation of mucin-type protein O-glycosylation by the polypeptide GalNAc transferase family of glycosyltransferases

Mammalian mucin-type O-glycosylation is initiated by a large family of ～20 UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferases (ppGalNAc Ts) that transfer α-GalNAc from UDP-GalNAc to Ser and Thr residues of polypeptide acceptors. Characterizing the peptide substrate specificity of each isoform is critical to understanding their properties, biological roles, and significance. Presently, only the specificities of ppGalNAc T1, T2, and T10 and the fly orthologues of T1 and T2 have been systematically characterized utilizing random peptide substrates. We now extend these studies to ppGalNAc T3, T5, and T12, transferases variously associated with human disease. Our results reveal several common features; the most striking is the similar pattern of enhancements for the three residues C-terminal to the site of glycosylation for those transferases that contain a common conserved Trp. In contrast, residues N-terminal to the site of glycosylation show a wide range of isoform-specific enhancements, with elevated preferences for Pro, Val, and Tyr being the most common at the -1 position. Further analysis reveals that the ratio of positive (Arg, Lys, and His) to negative (Asp and Glu) charged residue enhancements varied among transferases, thus further modulating substrate preference in an isoform-specific manner. By utilizing the obtained transferase-specific preferences, the glycosylation patterns of the ppGalNAc Ts against a series of peptide substrates could roughly be reproduced, demonstrating the potential for predicting isoform-specific glycosylation. We conclude that each ppGalNAc T isoform may be uniquely sensitive to peptide sequence and overall charge, which together dictates the substrate sites that will be glycosylated.     

The influence of flanking sequences on O-glycosylation

The influence of flanking sequences on O-glycosylation of serine and threonine residues was explored by comparison of known acceptor sites. Positions -6, -1 and +3 relative to the site were identified as particularly significant. To test the hypothesis that O-glycosylation could be affected by amino acid sequence, a series of test peptides was made containing substitutions at the sensitive positions. In vitro glycosylation of the peptides confirmed that the acceptor status of threonine was markedly influenced by the residues present at positions -6, -1 and +3. Circular dichroism indicated that peptides which had random structure were glycosylated, except when they contained a charged residue at position -1.     

The degradation of glycoproteins with lithium borohydride. Isolation and analysis of O-glycopeptides with reduced C-terminal amino acid residue

By the example of fetuin and a blood-group-specific mucin from porcine stomach, we showed that, under conditions of reductive degradation of glycoproteins with LiBH4-LiOH in 70% aqueous tert-butyl alcohol, the reduction and cleavage of amide bonds occur much faster than the simultaneous beta-elimination of carbohydrate chains O-linked with Ser and Thr residues of the peptide chain. The major degradation products containing the O-linked glycans are the O-glycosylated derivatives of 2-aminopropane-1,3-diol and 2-aminobutane-1,3-diol (the products of reduction of glycosylated Ser and Thr) and the glycopeptides containing 2-4 amino acid residues with reduced C-terminal amino acid. Seventeen homogeneous O-glycopeptides were isolated from the fetuin degradation products by ion-exchange and reversed-phase HPLC. Their structures were determined by MALDI-TOF mass spectrometry and by analyses for amino acids, amino alcohols, and carbohydrates. The application of the reaction for characterization of O-glycans and localization of O-glycosylation sites in O- and N,O-glycoproteins is discussed.     

Glycosylation sites identified by detection of glycosylated amino acids released from Edman degradation: the identification of Xaa-Pro-Xaa-Xaa as a motif for Thr-O-glycosylation

Here we report the use of automated Edman degradation of covalently linked glycopeptides to identify positively the sites of O- and N-glycosylation. The O-glycosidic linkage of carbohydrate to the hydroxy amino acids Ser and Thr is a major form of post-translational modification. However, unlike Asn-linked glycosylation, which is identified by the consensus sequence Asn-Xaa-Thr/Ser, no simple motif conferring O-linkage to Thr and Ser has been described. After sequencing glycopeptides derived from two cell surface glycoproteins, a Thr-O-glycosylation motif of Xaa-Pro-Xaa-Xaa, where at least one Xaa = Thr(Sac), has been defined. This motif predicts the site(s) of Pro- associated Thr-O-glycosylation in O-glycosylated proteins, although it is clear that there are also other forms of Thr-O-glycosylation not associated with Pro.     

Isolation and characterization of two biologically active O-glycosylated forms of human parathyroid hormone produced in Saccharomyces cerevisiae. Identification of a new motif for O-glycosylation.

Expression and secretion of human parathyroid hormone in Saccharomyces cerevisiae were achieved by fusing a cDNA encoding the mature human parathyroid hormone (hPTH) to the preproregion of the yeast mating factor alpha. Purified hPTH from yeast-culture medium was found to contain, in addition to the native unglycosylated form, two mannosylated variants with different molecular masses. The three hPTH forms were processed identically, resulting in the same 84 amino acid polypeptides with amino acid sequences identical to the native hormone. In both the O-glycosylated forms that were separated by isocratic reverse-phase HPLC, two mannose-linked residues were localized to Thr79. In addition, the most glycosylated form showed a heterogeneous modification of three, four or five mannosyl residues linked at Ser66. Lysine is N-terminally located to Ser66 and probably stimulates this glycosylation, which introduces a possible new motif for O-glycosylation in yeast. The two glycosylated forms of hPTH had similar biological activity which was identical to the native form of hPTH in a hormone-sensitive adenylate cyclase assay in bone sarcoma cells. Thus, a C-terminal O-glycosylation of hPTH with up to seven mannosyl residues/molecule did not affect the biological activity of the hormone, making possible production of hPTH with potential different pharmacokinetic properties.     

Building pathway clusters from Random Forests classification using class votes

Background

As one of the most common protein post-translational modifications, glycosylation is involved in a variety of important biological processes. Computational identification of glycosylation sites in protein sequences becomes increasingly important in the post-genomic era. A new encoding scheme was employed to improve the prediction of mucin-type O-glycosylation sites in mammalian proteins.

Results

A new protein bioinformatics tool, CKSAAP_OGlySite, was developed to predict mucin-type O-glycosylation serine/threonine (S/T) sites in mammalian proteins. Using the composition of k-spaced amino acid pairs (CKSAAP) based encoding scheme, the proposed method was trained and tested in a new and stringent O-glycosylation dataset with the assistance of Support Vector Machine (SVM). When the ratio of O-glycosylation to non-glycosylation sites in training datasets was set as 1:1, 10-fold cross-validation tests showed that the proposed method yielded a high accuracy of 83.1% and 81.4% in predicting O-glycosylated S and T sites, respectively. Based on the same datasets, CKSAAP_OGlySite resulted in a higher accuracy than the conventional binary encoding based method (about +5.0%). When trained and tested in 1:5 datasets, the CKSAAP encoding showed a more significant improvement than the binary encoding. We also merged the training datasets of S and T sites and integrated the prediction of S and T sites into one single predictor (i.e. S+T predictor). Either in 1:1 or 1:5 datasets, the performance of this S+T predictor was always slightly better than those predictors where S and T sites were independently predicted, suggesting that the molecular recognition of O-glycosylated S/T sites seems to be similar and the increase of the S+T predictor's accuracy may be a result of expanded training datasets. Moreover, CKSAAP_OGlySite was also shown to have better performance when benchmarked against two existing predictors.

Conclusion

Because of CKSAAP encoding's ability of reflecting characteristics of the sequences surrounding mucin-type O-glycosylation sites, CKSAAP_ OGlySite has been proved more powerful than the conventional binary encoding based method. This suggests that it can be used as a competitive mucin-type O-glycosylation site predictor to the biological community. CKSAAP_OGlySite is now available at http://bioinformatics.cau.edu.cn/zzd_lab/CKSAAP_OGlySite/.     

Glycosylation motifs that direct arabinogalactan addition to arabinogalactan-proteins

Hydroxyproline (Hyp)-rich glycoproteins (HRGPs) participate in all aspects of plant growth and development. HRGPs are generally highly O-glycosylated through the Hyp residues, which means carbohydrates help define the interactive molecular surface and, hence, HRGP function. The Hyp contiguity hypothesis predicts that contiguous Hyp residues are sites of HRGP arabinosylation, whereas clustered noncontiguous Hyp residues are sites of galactosylation, giving rise to the arabinogalactan heteropolysaccharides that characterize the arabinogalactan-proteins. Early tests of the hypothesis using synthetic genes encoding only clustered noncontiguous Hyp in the sequence (serine [Ser]-Hyp-Ser-Hyp)(n) or contiguous Hyp in the series (Ser-Hyp-Hyp)(n) and (Ser-Hyp-Hyp-Hyp-Hyp)(n) confirmed that arabinogalactan polysaccharide was added only to noncontiguous Hyp, whereas arabinosylation occurred on contiguous Hyp. Here, we extended our tests of the codes that direct arabinogalactan polysaccharide addition to Hyp by building genes encoding the repetitive sequences (alanine [Ala]-proline [Pro]-Ala-Pro)(n), (threonine [Thr]-Pro-Thr-Pro)(n), and (valine [Val]-Pro-Val-Pro)(n), and expressing them in tobacco (Nicotiana tabacum) Bright-Yellow 2 cells as fusion proteins with green fluorescent protein. All of the Pro residues in the (Ala-Pro-Ala-Pro)(n) fusion protein were hydroxylated and consistent with the hypothesis that every Hyp residue was glycosylated with arabinogalactan polysaccharide. In contrast, 20% to 30% of Pro residues remained non-hydroxylated in the (Thr-Pro-Thr-Pro)(n), and (Val-Pro-Val-Pro)(n) fusion proteins. Furthermore, although 50% to 60% of the Hyp residues were glycosylated with arabinogalactan polysaccharide, some remained non-glycosylated or were arabinosylated. These results suggest that the amino acid side chains of flanking residues influence the extent of Pro hydroxylation and Hyp glycosylation and may explain why isolated noncontiguous Hyp in extensins do not acquire an arabinogalactan polysaccharide but are arabinosylated or remain non-glycosylated.     

A systematic study of site-specific GalNAc-type O-glycosylation modulating proprotein convertase processing

Site-specific GalNAc-type O-glycosylation is emerging as an important co-regulator of proprotein convertase (PC) processing of proteins. PC processing is crucial in regulating many fundamental biological pathways and O-glycans in or immediately adjacent to processing sites may affect recognition and function of PCs. Thus, we previously demonstrated that deficiency in site-specific O-glycosylation in a PC site of the fibroblast growth factor, FGF23, resulted in marked reduction in secretion of active unprocessed FGF23, which cause familial tumoral calcinosis and hyperostosis hyperphosphatemia. GalNAc-type O-glycosylation is found on serine and threonine amino acids and up to 20 distinct polypeptide GalNAc transferases catalyze the first addition of GalNAc to proteins making this step the most complex and differentially regulated steps in protein glycosylation. There is no reliable prediction model for O-glycosylation especially of isolated sites, but serine and to a lesser extent threonine residues are frequently found adjacent to PC processing sites. In the present study we used in vitro enzyme assays and ex vivo cell models to systematically address the boundaries of the region within site-specific O-glycosylation affect PC processing. The results demonstrate that O-glycans within at least ±3 residues of the RXXR furin cleavage site may affect PC processing suggesting that site-specific O-glycosylation is a major co-regulator of PC processing.     

Prediction, conservation analysis, and structural characterization of mammalian mucin-type O-glycosylation sites

O-GalNAc-glycosylation is one of the main types of glycosylation in mammalian cells. No consensus recognition sequence for the O-glycosyltransferases is known, making prediction methods necessary to bridge the gap between the large number of known protein sequences and the small number of proteins experimentally investigated with regard to glycosylation status. From O-GLYCBASE a total of 86 mammalian proteins experimentally investigated for in vivo O-GalNAc sites were extracted. Mammalian protein homolog comparisons showed that a glycosylated serine or threonine is less likely to be precisely conserved than a nonglycosylated one. The Protein Data Bank was analyzed for structural information, and 12 glycosylated structures were obtained. All positive sites were found in coil or turn regions. A method for predicting the location for mucin-type glycosylation sites was trained using a neural network approach. The best overall network used as input amino acid composition, averaged surface accessibility predictions together with substitution matrix profile encoding of the sequence. To improve prediction on isolated (single) sites, networks were trained on isolated sites only. The final method combines predictions from the best overall network and the best isolated site network; this prediction method correctly predicted 76% of the glycosylated residues and 93% of the nonglycosylated residues. NetOGlyc 3.1 can predict sites for completely new proteins without losing its performance. The fact that the sites could be predicted from averaged properties together with the fact that glycosylation sites are not precisely conserved indicates that mucin-type glycosylation in most cases is a bulk property and not a very site-specific one. NetOGlyc 3.1 is made available at www.cbs.dtu.dk/services/netoglyc.     

Concepts and Principles of O-Linked Glycosylation

The biosynthesis, structures, and functions of O-glycosylation, as a complex posttranslational event, is reviewed and compared for the various types of O-glycans. Mucin-type O-glycosylation is initiated by tissue-specific addition of a GalNAc-residue to a serine or a threonine of the fully folded protein. This event is dependent on the primary, secondary, and tertiary structure of the glycoprotein. Further elongation and termination by specific transferases is highly regulated. We also describe some of the physical and biological properties that O-glycosylation confers on the protein to which the sugars are attached. These include providing the basis for rigid conformations and for protein stability. Clustering of O-glycans in Ser/Thr(/Pro)-rich domains allows glycan determinants such as sialyl Lewis X to be presented as multivalent ligands, essential for functional recognition. An additional level of regulation, imposed by exon shuffling and alternative splicing of mRNA, results in the expression of proteins that differ only by the presence or absence of Ser/Thr(/Pro)-rich domains. These domains may serve as protease-resistant spacers in cell surface glycoproteins. Further biological roles for O-glycosylation discussed include the role of isolated mucin-type O-glycans in recognition events (e.g., during fertilization and in the immune response) and in the modulation of the activity of enzymes and signaling molecules. In some cases, the O-linked oligosac-charides are necessary for glycoprotein expression and processing. In contrast to the more common mucin-type O-glycosylation, some specific types of O-glycosylation, such as the O-linked attachment of fucose and glucose, are sequon dependent. The reversible attachment of O-linked GlcNAc to cytoplasmic and nuclear proteins is thought to play a regulatory role in protein function. The recent development of novel technologies for glycan analysis promises to yield new insights in the factors that determine site occupancy, structure-function relationship, and the contribution of O-linked sugars to physiological and pathological processes. These include diseases where one or more of the O-glycan processing enzymes are aberrantly regulated or deficient, such as HEMPAS and cancer.     

Unusual glycosylation of proteins: Beyond the universal sequon and other amino acids

BackgroundGlycosylation of proteins is the most common, multifaceted co- and post-translational modification responsible for many biological processes and cellular functions. Significant alterations and aberrations of these processes are related to various pathological conditions, and often turn out to be disease biomarkers. Conventional N-glycosylation occurs through the recognition of the consensus sequon, asparagine (Asn)-X-serine (Ser)/threonine (Thr), where X is any amino acid except for proline, with N-acetylglucosamine (GlcNAc) as the first glycosidic linkage. Usually, O-glycosylation adds a glycan to the hydroxyl group of Ser or Thr beginning with N-acetylgalactosamine (GalNAc).Scope of reviewProtein glycosylation is further governed by additional diversifications in sequon and structure, which are yet to be fully explored. This review mainly focuses on the occurrence of N-glycosylation in non-consensus motifs, where Ser/Thr at the + 2 position is substituted by other amino acids. Additionally, N-glycosylation is also observed in other amide/amine group-containing amino acids. Similarly, O-glycosylation occurs at hydroxyl group-containing amino acids other than serine/threonine. The neighbouring amino acids and local structural features around the potential glycosylation site also play a significant role in determining the extent of glycosylation. All of these phenomena that yield glycosylation at the atypical sites are reported in a variety of biological systems, including different pathological conditions.Conclusion and SignificanceTherefore, the discovery of more novel sequence patterns for N- and O-glycosylation may help in understanding the functions of complex biological processes and cellular functions. Taken together, all these information provided in this review would be helpful for the biological readers.     

The influence of flanking sequence on the O-glycosylation of threonine in vitro.

To investigate the influence of flanking amino acid sequence on the O-glycosylation of a single threonine residue in vitro, we have examined a series of 52 related peptides. The substrates were based upon a sequence from human von Willebrand factor which is known to be glycosylated in vivo (-6PHMAQVTVGPGL+5). Each residue of the parent peptide was substituted, in turn, with isoleucine, alanine, proline, glutamic acid, or arginine. Peptides were glycosylated using a UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase purified 15,000-fold from bovine colostrum by chromatography on DEAE-Sephacel, SP-Sephadex, Sephacryl S-300, Affi-Gel Blue, and 5-mercuri-UDP-GalNAc thiopropyl-Sepharose. Single amino acid changes in the sequences flanking the threonine could profoundly alter the glycosylation of the substrate peptides. Substitution of any amino acid tested at positions +3, -3, and -2 markedly decreased O-glycosylation, as did the presence of a charged residue at position -1. The substitution of amino acids at the other positions of the peptide substrate had little effect on the incorporation of GalNAc. Statistical analysis of sequences flanking known glycosylated threonine and serine residues suggests that they should be glycosylated with equal efficiency in the same sequence context (O'Connell et al., 1991). However, the bovine colostrum transferase failed to glycosylate a peptide derived from human erythropoietin which contains a serine that is glycosylated in vivo (-5PPDAASAAPLR+5). When a threonine was substituted for the serine in this peptide (-5PPDAATAAPLR+5), the substrate proved to be an excellent acceptor of GalNAc. These observations indicate that although flanking amino acid sequence is important for the O-glycosylation of specific hydroxyamino acids, discrete threonine- and serine-specific transferases may exist.     

The catalytic and lectin domains of UDP-GalNAc:polypeptide alpha-N-Acetylgalactosaminyltransferase function in concert to direct glycosylation site selection

UDP-GalNAc:polypeptide alpha-N-Acetylgalactosaminyltransferases (ppGalNAcTs), a family (EC 2.4.1.41) of enzymes that initiate mucin-type O-glycosylation, are structurally composed of a catalytic domain and a lectin domain. Previous studies have suggested that the lectin domain modulates the glycosylation of glycopeptide substrates and may underlie the strict glycopeptide specificity of some isoforms (ppGalNAcT-7 and -10). Using a set of synthetic peptides and glycopeptides based upon the sequence of the mucin, MUC5AC, we have examined the activity and glycosylation site preference of lectin domain deletion and exchange constructs of the peptide/glycopeptide transferase ppGalNAcT-2 (hT2) and the glycopeptide transferase ppGalNAcT-10 (hT10). We demonstrate that the lectin domain of hT2 directs glycosylation site selection for glycopeptide substrates. Pre-steady-state kinetic measurements show that this effect is attributable to two mechanisms, either lectin domain-aided substrate binding or lectin domain-aided product release following glycosylation. We find that glycosylation of peptide substrates by hT10 requires binding of existing GalNAcs on the substrate to either its catalytic or lectin domain, thereby resulting in its apparent strict glycopeptide specificity. These results highlight the existence of two modes of site selection used by these ppGalNAcTs: local sequence recognition by the catalytic domain and the concerted recognition of distal sites of prior glycosylation together with local sequence binding mediated, respectively, by the lectin and catalytic domains. The latter mode may facilitate the glycosylation of serine or threonine residues, which occur in sequence contexts that would not be efficiently glycosylated by the catalytic domain alone. Local sequence recognition by the catalytic domain differs between hT2 and hT10 in that hT10 requires a pre-existing GalNAc residue while hT2 does not.     

Site directed processing: Role of amino acid sequences and glycosylation of acceptor glycopeptides in the assembly of extended mucin type O-glycan core 2

Background

The assembly of Ser/Thr-linked O-glycans of mucins with core 2 structures is initiated by polypeptide GalNAc-transferase (ppGalNAc-T), followed by the action of core 1 β3-Gal-transferase (C1GalT) and core 2 β6-GlcNAc-transferase (C2GnT). β4-Gal-transferase (β4GalT) extends core 2 and forms the backbone structure for biologically important epitopes. O-glycan structures are often abnormal in chronic diseases. The goal of this work is to determine if the activity and specificity of these enzymes are directed by the sequences and glycosylation of substrates.

Methods

We studied the specificities of four enzymes that synthesize extended O-glycan core 2 using as acceptor substrates synthetic mucin derived peptides and glycopeptides, substituted with GalNAc or O-glycan core structures 1, 2, 3, 4 and 6.

Results

Specific Thr residues were found to be preferred sites for the addition of GalNAc, and Pro in the + 3 position was found to especially enhance primary glycosylation. An inverse relationship was found between the size of adjacent glycans and the rate of GalNAc addition. All four enzymes could distinguish between substrates having different amino acid sequences and O-glycosylated sites. A short glycopeptide Galβ1–3GalNAcα-TAGV was identified as an efficient C2GnT substrate.

Conclusions

The activities of four enzymes assembling the extended core 2 structure are affected by the amino acid sequence and presence of carbohydrates on nearby residues in acceptor glycopeptides. In particular, the sequences and O-glycosylation patterns direct the addition of the first and second sugar residues by ppGalNAc-T and C1GalT which act in a site directed fashion.

General significance

Knowledge of site directed processing enhances our understanding of the control of O-glycosylation in normal cells and in disease.     

A serial lectin approach to the mucin-type O-glycoproteome of Drosophila melanogaster S2 cells

Identification of mucin-type O-glycosylated proteins with known functions in model organisms like Drosophila could provide keys to elucidate functions of the O-glycan moiety and proteomic analyses of O-glycoproteins in higher eukaryotes remain a challenge due to structural heterogeneity and a lack of efficient tools for their specific isolation. Here we report a strategy to evaluate the O-glycosylation potential of the embryonal hemocyte-like Drosophila Schneider 2 (S2) cell line by expression of recombinant glycosylation probes derived from tandem repeats of the human mucin MUC1 or of the Drosophila salivary gland protein Sgs1. We obtained evidence that mucin-type O-glycosylation in S2 cells grown under serum-free conditions is restricted to the Tn-antigen (GalNAcalpha-Ser/Thr) and the T-antigen (Galbeta1-3GalNAcalpha-Ser/Thr) and this structural homogeneity enables unique glycoproteomic strategies. We present a label-free strategy for the isolation, profiling and analysis of O-glycosylated proteins consisting of serial lectin affinity capture, 2-DE-based glycoprotein analysis by O-glycan specific mAbs and protein identification by MALDI-MS. Protein identity and O-glycosylation was confirmed by ESI-MS/MS with detection of diagnostic sugar oxonium-ion fragments. Using this strategy, we established 2-D reference maps and identified 21 secreted and intracellular mucin-type O-glycoproteins. Our results show that Drosophila S2 cells express O-glycoproteins involved in a wide range of biological functions including proteins of the extracellular matrix (Laminin gamma-chain, Peroxidasin and Glutactin), pathogen recognition proteins (Gnbp1), stress response proteins (Glycoprotein 93), secreted proteases (Matrix-metalloprotease 1 and various trypsin-like serine proteases), protease inhibitors (Serpin 27 A) and proteins of unknown function.     

The location and characterisation of the O-linked glycans of the human insulin receptor

O-linked glycosylation is a post-translational and post-folding event involving exposed S/T residues at beta-turns or in regions with extended conformation. O-linked sites are difficult to predict from sequence analyses compared to N-linked sites. Here we compare the results of chemical analyses of isolated glycopeptides with the prediction using the neural network prediction method NetOGlyc3.1, a procedure that has been reported to correctly predict 76% of O-glycosylated residues in proteins. Using the heavily glycosylated human insulin receptor as the test protein six sites of mucin-type O-glycosylation were found at residues T744, T749, S757, S758, T759, and T763 compared to the three sites (T759 and T763- correctly, T756- incorrectly) predicted by the neural network method. These six sites occur in a 20 residue segment that begins nine residues downstream from the start of the insulin receptor beta-chain. This region which also includes N-linked glycosylation sites at N742 and N755, is predicted to lack secondary structure and is followed by residues 765-770, the known linear epitope for the monoclonal antibody 18-44.     

Role of peptide sequence and neighboring residue glycosylation on the substrate specificity of the uridine 5'-diphosphate-alpha-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyl transferases T1 and T2: kinetic modeling of the porcine and canine submaxillary gland mucin tandem repeats

A large family of uridine 5'-diphosphate (UDP)-alpha-N-acetylgalactosamine (GalNAc):polypeptide N-acetylgalactosaminyl transferases (ppGalNAc Ts) initiates mucin-type O-glycan biosynthesis at serine and threonine. The peptide substrate specificities of individual family members are not well characterized or understood, leaving an inability to rationally predict or comprehend sites of O-glycosylation. Recently, a kinetic modeling approach demonstrated neighboring residue glycosylation as a major factor modulating the O-glycosylation of the porcine submaxillary gland mucin 81 residue tandem repeat by ppGalNAc T1 and T2 [Gerken et al. (2002) J. Biol. Chem. 277, 49850-49862]. To confirm the general applicability of this model and its parameters, the ppGalNAc T1 and T2 glycosylation kinetics of the 80+ residue tandem repeat from the canine submaxillary gland mucin was obtained and characterized. To reproduce the glycosylation patterns of both mucins (comprising 50+ serine/threonine residues), specific effects of neighboring peptide sequence, in addition to the previously described effects of neighboring residue glycosylation, were required of the model. Differences in specificity of the two transferases were defined by their sensitivities to neighboring proline and nonglycosylated hydroxyamino acid residues, from which a ppGalNAc T2 motif was identified. Importantly, the model can approximate the previously reported ppGalNAc T2 glycosylation kinetics of the IgA1 hinge domain peptide [Iwasaki, et al. (2003) J. Biol. Chem. 278, 5613-5621], further validating both the approach and the ppGalNAc T2 positional weighting parameters. The characterization of ppGalNAc transferase specificity by this approach may prove useful for the search for isoform-specific substrates, the creation of isoform-specific inhibitors, and the prediction of mucin-type O-glycosylation sites.     

Conformation analysis of the N-glycosylation site Asn-X-Thr/Ser in glycoproteins

Theoretical conformational analysis of oligopeptides CH3CO-Asn-X-Thr-NHCH3 (X = Gly, Ala, Pro), modelling N-glycosylation site, and their glycosylated derivatives CH3CO-(GlcNAc beta 1-4GlcNAc beta 1) Asn-X-Thr-NHCH3 has been carried out. Active conformations of the site are found, corresponding to structural prerequisities of N-glycosylation: Asn residue's position in beta-turn and hydrogen bond formation between side chains of Asn and Thr/Ser residues. In this case the L conformation of the central residue X is most probable. Since Pro residue does not possess this conformation, sequences with X = Pro are not glycosylated. It is shown that glycosylation of the above-mentioned sites is accompanied by reorientation of the Asn residue's side chains.     

MAPRes: Mining association patterns among preferred amino acid residues in the vicinity of amino acids targeted for post-translational modifications

Post-translational modification (PTM) of a protein is an important event in regulating cellular functions. An algorithm, MAPRes, has been developed for mining associations among PTM sites and the preferred amino acids in their vicinity. The algorithm has been implemented to O-glycosylation and O-phosphorylation data (phosphorylated/glycosylated Ser/Thr/Tyr). The association patterns mined by MAPRes demonstrate significant correlations and the results are in conformity with the existing methods. These association rules/patterns will be helpful in predicting the sequences/motifs involved for specific PTMs in proteins.