INTRODUCTION

Mucin glycoproteins are heavily O-glycosylated glycoproteins secreted by higher organisms that serve vital roles, protecting and lubricating epithelial cell surfaces from biological, chemical, and mechanical insult. Mucins and mucin-like molecules attached to membrane and cell surfaces play additional important roles by modulating for example immune response, inflammation, and tumorigenesis (1-5). The O-glycosylated domains of mucins and mucin-like glycoproteins typically contain 50-80% carbohydrate and possess expanded conformations. These regions typically contain high Ser and Thr contents, commonly composed of polypeptide tandem repeats containing clusters of Ser and Thr residues. It has been demonstrated, in the case of mucins, that the O-linked oligosaccharide side chains, attached via -N-acetylgalactosamine (GalNAc)¹ to Ser and Thr, are solely responsible for their 3-fold expanded peptide chain dimensions (6). Chemical and NMR studies indicate that on average 75% or more of the Ser and Thr residues in mucins are glycosylated (7, 8). Little, however, is known of the actual distribution of the carbohydrate in these clusters along the mucin polypeptide core. In addition, it is unknown whether the distribution of oligosaccharides along the peptide core is random or whether specific Ser/Thr residues are preferentially glycosylated over others. Obtaining this information by peptide mapping approaches would be a significant analytical undertaking due to the vast array of glycopeptides with different glycosylation patterns and oligosaccharide structures that would be needed to be quantitatively isolated and characterized.

Several predictive methods, based on the analysis of reported O-glycosylation sites compiled from protein data bases, are available for estimating the relative propensity for a given Ser or Thr to be glycosylated (9-11). Application of these predictive methods to the highly glycosylated mucins may not be fully valid because mucins and mucin-like molecules were not a significant component of the "training" data sets, which were dominated by globular glycoproteins, and where presented, the data on mucins and mucin-like glycoproteins glycosylation are commonly incomplete or in error. Furthermore, the propensity for O-glycosylation of Ser and Thr residues at the surface of globular glycoproteins may vary considerably from those found in mucin-like glycoproteins, which presumably serve different structural functions.

In vitro O-glycosylation studies using synthetic peptide acceptors have also been utilized to determine the propensity for a given Ser and Thr to be O-glycosylated (12-19). Unfortunately, the isolated peptide -N-acetylgalactosaminyltransferases give different extents of Ser/Thr glycosylation on peptide substrates compared with that observed in vivo (14, 20). This may be the result of altered enzyme specificity as a result of inappropriate solution conditions, absence of cofactors, the presence of more than one transferase (21-23), or the result of the artifacts resulting from the use of relatively small peptides containing charged N and C termini. Only by characterizing and quantifying the specific in vivo glycosylation of native and/or expressed recombinant proteins and peptides (20) is it likely that valid and useful data will be obtained for determining the true in vivo transferase specificities.

To begin to address the structural effects of O-glycosylation and to determine the extent that mucin O-glycosylation is modulated in vivo in a site-specific manner, we have undertaken the isolation and characterization of the porcine submaxillary gland mucin (PSM)-glycosylated tandem repeat. We have determined the glycosylation pattern of the isolated 81-residue tandem repeat and have isolated and characterized several smaller PSM tandem repeat-derived glycopeptides (24). This work provides the first detailed analysis of the glycosylation pattern of a mucin and suggests that mucin glycosylation is modulated by peptide sequence but not entirely as expected by the existing O-glycosylation prediction algorithms. Based on the observed glycosylation pattern a model for the GalNAc transferase peptide binding site has been proposed.

EXPERIMENTAL PROCEDURES

Materials

-GalNAc-Thr and -GalNAc-Ser were a kind gift of R. Koganti, Biomera Inc. Edmonton, Alberta, Canada. Except where noted, all chemicals and enzyme reagents were obtained from Sigma or Fisher.

Methods

Porcine submaxillary gland mucin was obtained from frozen porcine submaxillary glands, in gram quantities, as described by Shogren et al. (25).

PSM was reduced and carboxymethylated by the methods of Gupta and Jentoft (32) giving R-PSM. R-PSM (1 g/50 ml) was digested with L-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin (15 mg/1 g R-PSM) (Worthington) overnight at 37 °C in 50 mM ammonium bicarbonate, pH 8.3. A second aliquot of trypsin was added to ensure complete digestion and incubated 5-8 h. Toluene was added to both incubation solutions to prevent microbial growth. After exhaustive dialysis and the removal of insoluble debris by centrifugation, trypsinized R-PSM (TR-PSM) was lyophilized.

Lyophilized and dissector-dried TR-PSM (~0.75 g in 75-ml Teflon screw-cap tubes) was reacted for 6-16 h with a TFMSA (50 g) anisole (15 ml) (Aldrich) mixture at 0 °C following the approach of Gerken et al. (26, see also Ref. 27). To reduce heating effects, both the reagents and lyophilized TR-PSM were chilled in dry ice/ethanol prior to their mixing. After incubation with occasional vigorous shaking, the reaction was again chilled, and 1 volume of cold anhydrous diethyl ether was added. This mixture was slowly added to 125 ml of a frozen slush of 60% pyridine, after which the solution was warmed to room temperature and extracted with ether. The aqueous phase containing the partially deglycosylated TR-PSM (TTR-PSM) was dialyzed exhaustively and lyophilized.

Low molecular weight non-glycosylated peptides were separated from the glycosylated tandem repeat subunits by gel filtration chromatography on Sephacryl S200 (Pharmacia Biotech, Uppsala, Sweden) (column dimensions 5 × 55 cm, 7-ml fraction volumes) eluted with 50 mM ammonium bicarbonate buffer. Glycoprotein content was monitored by periodic acid-Schiff reagent (28), absorbance at 555 nm, and protein monitored by the absorbance at 220 nm.

The high molecular weight carbohydrate-containing fraction eluting near the void volume of the S200 column was lyophilized and treated a second time with L-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin (10 mg/g TTR-PSM) using the conditions described for the initial trypsin treatment. The digested TTR-PSM (TTTR-PSM) was fractionated on S200, and the PSM tandem repeats were isolated as the major included glycopeptide fraction (TTTR-PSM-T3) and lyophilized.

The TTTR-PSM-T3 tandem repeat (30 mg) in 5 ml of 25 mM ammonium bicarbonate, pH 7.8, was digested with 1 mg of protease Glu-C (Boehringer Mannheim) for 20 h at 25 °C. After a second addition of Glu-C and further digestion, the mixture was fractionated by S200 chromatography. The major glycopeptide peak (GTTTR-PSM) was separated into the major N- and C-terminal tandem repeat peptides on reverse phase HPLC.

The TTTR-PSM-T3 tandem repeat and Glu-C-digested repeat (GTTTR-PSM) were further purified by reverse phase HPLC chromatography on a 0.46 × 15 cm C18 ODSII column (Alltech Associates Inc., Dearfield IL) using 0.05% trifluoroacetic acid, water/acetonitrile gradients as described in the figure legend. All isolations were performed on a Varian 5000 HPLC system (Varian Associates, Walnut Creek CA) equipped with a Schamitsu UV/VIS detector.

Pulsed liquid phase Edman degradation amino acid sequencing of the isolated PSM tandem repeats and Glu-C-derived glycopeptide was performed on either an Applied Biosystems 477A or Applied Biosystems Procise 494 protein sequencer (Perkin-Elmer) typically using standard manufacturer recommended pulsed-liquid cycles (24). Samples of 2000-5000 pmol were dried on trifluoroacetic acid washed glass fiber filters (ABI number 401111) spotted with 1.5 mg of BioBrene Plus (ABI 400385). Amino acid phenylthiohydantoin (PTH) derivatives were chromatographed on standard ABI 5-µm C18 PTH columns using the Fast Normal 1 gradient program and were monitored by the absorbance at 269 nm. The PTH-Thr/Ser-O-GalNAc derivatives were found to elute as two diastereotopic peaks at unique positions in the chromatogram (24). Due to increased peak broadening and changes in elution position as the PTH columns age, some variability and overlap of the glycosylated PTH derivatives with Ser, Thr, Gly, and Asp PTH derivatives were commonly observed. Since the ratio of the areas of the diastereotopic PTH-Ser/Thr-O-GalNAc derivatives was found to be relatively constant (typically 45/55 for Thr), the extent of glycosylation usually could be determined by the use of simple algebra. Modifications in the HPLC gradient were found to produce only marginal improvement in the separation of the PTH-Ser/Thr-GalNAc derivatives from the elution positions of neighboring PTH-derivatives. Prior to data analysis, long term cycle preview and lag for each PTH-derivative was eliminated by a base-line subtraction approach. This was performed by subtracting the 5-cycle minimum value running average across the entire sequencing run for each peak. Due to the length of the peptides and high content of similar amino acids (i.e. Gly, Ser, Thr, and Ala), no attempts were made to include quantifications for adjacent residue cycle lag or preview. Response factors for the PTH-Ser/Thr-O-GalNAc derivatives were obtained from shorter glycopeptide sequencing experiments (24) and were found to be similar to those of Ser and Thr. Response values for Ser, Thr, and their glycosylated PTH-derivatives were further adjusted for each sequencing run to obtain consistent picomole yields relative to the non-Ser/Thr residues.

The PSM tandem repeat sequence was analyzed for potential O-glycosylation sites using software kindly provided by Dr. A. Elhammer (9) and by using the E-mail NetOglyc server of Hansen et al. (10). The sequence coupled vector projection predictions were kindly performed by Dr. K. Chou (11). Peptide secondary structure predictions were performed by the SOPM internet server (29). Predictions were performed for at least 10 residues beyond the tandem repeat N and C-terminal boundaries to eliminate end effects.

Peptides were modeled using the Biopolymer module of InsightII (MSI Inc. formally Biosym Technologies, San Diego CA).

RESULTS

The structure of porcine submaxillary mucin (PSM) polypeptide based on the nucleotide sequencing of Timpte et al. (30) and Eckhardt et al. (31) is shown in Fig. 1A. The mucin's structure is dominated by the presence of highly O-glycosylated, multiple repeating 81-residue tandem repeats. These repeats make up the vast majority of the mucin's amino acid sequence and represent the major glycosylation sites in PSM.

There are two potential trypsin cleavage sites, Arg-Ile and Arg-Pro, in each PSM tandem repeat. The Arg-Pro site, however, is expected to be inactive; therefore, trypsin treatment is expected to yield single copies of the 81-residue tandem repeat with the sequence given in Fig. 1B. Indeed, Timpte and co-workers (30) have shown that after full deglycosylation and digestion of the fully deglycosylated (apo) mucin with trypsin, this tryptic peptide is obtained. In contrast, Gupta and Jentoft (32) have shown that trypsin treatment of fully glycosylated, reduced and carboxymethylated mucin fails to yield single copies of the repeat and instead yields relatively high molecular weight glycopeptides composed of undigested tandem repeats. Apparently, the longer oligosaccharide side chains inhibit the digestion of the PSM polypeptide by trypsin, preventing the isolation of individual tandem repeats. The Sephacryl S200 gel filtration chromatogram of this species, TR-PSM, is given in Fig. 2A.

Only after quantitatively trimming the oligosaccharide side chains to the peptide-linked -GalNAc residue by mild TFMSA treatment (26-27), Fig. 2B, does the PSM glycopeptide core become susceptible to cleavage by trypsin, Fig. 2C, presumably releasing monomeric glycosylated tryptic tandem repeats (peak T3).² Mild TFMSA treatment does not appreciably degrade the mucin, since on gel filtration untreated and treated mucin (TR-PSM and TTR-PSM respectively) contain similar high molecular weight glycosylated peaks, Fig. 2, A and B. Integrations of the -carbon resonances of the ¹³C NMR spectra of native and TTR-PSM confirm that 96 to 97% of the glycosylated Ser and Thr residues of the native mucin retain intact unsubstituted -GalNAc residues after mild TFMSA treatment (data not shown, see Ref. 26).

The suspected monomeric glycosylated tryptic tandem repeat, pooled as indicated in Fig. 2C, gave a single sharp peak after rechromatography on S200 as shown in Fig. 2D (). On reverse phase HPLC, Fig. 3A, this peak gives a single somewhat broadened peak, representing the ensemble of heterogeneously glycosylated tandem repeats. This peak was pooled for amino acid sequencing and for subsequent digestion by Glu C (discussed below). Amino acid sequencing of this peak confirmed the isolation of the PSM tryptic tandem repeat.

Since each step in the above procedure typically yields a single major glycopeptide species that is pooled for the subsequent step, the obtained tandem repeat glycosylation pattern (see below) is expected to represent the majority of the PSM tandem repeats of the native mucin. After correcting for the different carbohydrate contents of native and TFMSA-modified PSM (26), it is calculated that between 40 and 50% (uncorrected for the nonspecific losses of material at each step) of the initial TR-PSM peptide is isolated in the pooled TTTR-PSM-T3 fraction. The possibility exists, however, that a subpopulation of differently glycosylated species may have been excluded, since the lower molecular weight regions of the glycopeptide peaks in Fig. 2, B and C, were not pooled. These lower molecular weight species are thought to represent nonspecific (protease and TFMSA) degradation products of the tandem repeat and other non-tandem repeat glycopeptides arising from the N- and C-terminal domains of PSM (see Fig. 1A). Evidence for nonspecific protease degradation arises from the observation that the use of non-L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin gives a T3 peak that is further broadened to lower molecular weight. By eliminating these lower molecular weight species we are able to reduce the background sequencing "noise," thereby permitting the sequencing of longer segments of the tandem repeat glycopeptide (discussed below). We believe, however, that these degradation products would not have significantly different glycosylation patterns compared with the intact tandem repeat.

The PSM tandem repeat contains three potential cleavage sites for endoproteinase Glu-C from Staphylococcus aureus V8 (cleaving at C terminus of Glu). Digestion of the tryptic PSM tandem repeat with Glu-C will therefore produce three glycopeptides of 40, 38, and 3 residues each, proceeding from the N to C terminus, respectively. We chose to isolate the 38-residue glycopeptide (residues 39-78) since its analysis would further help confirm the glycosylation pattern of the C-terminal half of the tandem repeat.

The HPLC-purified tryptic tandem repeat (Fig. 3A) was digested with Glu-C and fractionated on S200 chromatography, Fig. 2D. As shown in the figure the Glu-C digest () migrates at a lower molecular weight than undigested tryptic repeat (). The 40- and 38-residue glycopeptides are not expected to be resolved on S200; therefore, the production of a single sharp peak after Glu-C digestion indicates the repeat has been fully cleaved by the enzyme. The remaining 3-residue glycopeptide was not specifically isolated or identified but presumably appears near the included volume of the column approximately fractions 120-140.

On reverse phase HPLC the pooled product of the Glu-C digest (pooled as indicated in Fig. 2D, ) reveals a complex pattern comprised of two major broad peaks, labeled (GTTTR-PSM)-GII and (GTTTR-PSM)-GIII, as shown in Fig. 3B. Peak GII elutes at a lower acetonitrile content than the intact tryptic tandem repeat, whereas peak GIII elutes at a similar acetonitrile content as the intact tryptic tandem repeat. On the basis of the expected differences in hydrophobicities, the least hydrophobic fraction, GII, was tentatively assigned to the least hydrophobic glycopeptide 39-78 (containing 11 hydrophobic residues: Ala, Pro, Val, and Ile), and the more hydrophobic fraction, GIII, was tentatively assigned to glycopeptide 1-38 (containing 15 hydrophobic residues). Proton NMR spectroscopy, at 600 MHz, of each pooled fraction confirmed their identities based on their different amino acid compositions (data not shown). Fraction GTTTR-PSM-GII was unambiguously identified as residues 39-78 on the basis of its amino acid sequence, presented below.

Amino acid sequencing was performed on the reverse phase HPLC-purified tandem repeat glycopeptides, TTTR-PSM-T3 and GTTTR-PSM-GII. As described earlier (24, 34-35), unique elution patterns are observed for the PTH-derivatives of -GalNAc-Ser and -GalNAc-Thr as shown in Fig. 4A for authentic -GalNAc-Ser and -GalNAc-Thr. Each glycosylated PTH-derivative appears as a pair of peaks in the chromatogram (Fig. 4A) because the conversion reaction forming the amino acid-PTH-derivative produces diastereomers with different retention times. The -GalNAc-Ser-PTH-derivatives elute as an unresolved doublet, labeled S*+S**, early in the gradient near the position of PTH-Asp, and the -GalNAc-Thr-PTH diastereomers elute later in the gradient as resolved peaks T* and T**, near the positions of PTH-Ser and PTH-Thr, respectively. These peaks are readily identified in the sequencing chromatograms of glycopeptide TTTR-PSM-T3 for residues Ser², Ser⁶, and Thr²² as shown in Fig. 4, B-D. On the basis of the relative sizes of the glycosylated and nonglycosylated PTH-Ser/Thr derivatives in the TTTR-PSM-T3, it appears that Ser⁶ and Thr²² are more highly glycosylated than Ser², which seems to be poorly glycosylated.

Representative amino acid sequencing chromatograms of the -GalNAc-Ser and -GalNAc-Thr standards and TFMSA-treated PSM tryptic tandem repeats.

Sequencing chromatograms were quantified to obtain the residue-specific extent of glycosylation as illustrated in Figs. 5 and 6 for sequencing run 2 of TTTR-PSM-T3 tandem repeat glycopeptide. Fig. 5A displays the uncorrected area data for the Gly-PTH peak plotted as a function of sequence cycle. The figure shows a pronounced base-line curvature that is also observed for the other amino acid residues and glycosylated Ser/Thr (data not shown). Since the extent of base-line curvature correlated with the residue's percent mole fraction, we conclude that the curvature is due to the cumulative effects of cycle preview and lag and perhaps due to heterogeneous cleavage of the tandem repeat peptide. Since non-zero base lines will interfere with the accurate determination of the extent of glycosylation and with the sequence determination at high cycle numbers, we eliminated the curvature by the base-line subtraction approach described under "Experimental Procedures." The effectiveness of the base-line correction approach is shown in the corrected data for Gly, Ile, Val, and Ala of Fig. 5, C-F, and for glycosylated and nonglycosylated Ser and Thr of Fig. 6, A-D. Note that after base-line correction the sequence can be read well beyond residue 60 as demonstrated by the expanded plots of Fig. 5, D and F, and Fig. 6, B and D. A plot of the single residue picomoles recovered versus cycle number, Fig. 5B, indicates that we have achieved reasonable sequential residue quantification and recovery. An average apparent repetitive yield of 99% is obtained from the data in Fig. 5B.

Table I lists the calculated extent of Ser/Thr glycosylation obtained from the multiple sequencing of the PSM tryptic tandem repeat (TTTR-PSM-T3) and its C-terminal Glu-C glycopeptide (GTTTR-PSM-GII). Sequence determinations 1 and 2 represent data from the same sample sequenced on different instruments. Note the excellent agreement between the two sequencing experiments. Sequence determination 3 represents the tandem repeat obtained from a different PSM preparation. Again, the results of determination 3 are nearly indistinguishable from the results of sequence determinations 1 and 2. The glycosylation patterns of the C-terminal Glu-C tryptic tandem repeat glycopeptide, determinations 4 and 5, also are in good agreement with the data from the full tryptic tandem repeat.

Table I. Sequence-specific glycosylation patterns of the PSM tandem repeat The abbreviations used are: TTTR-PSM-T3, oligosaccharide trimmed PSM tryptic tandem repeat, residues 1-81, HPLC purified in Fig. 3A; GTTTR-PSM-GII, protease Glu-C-cleaved TTTR-PSM-T3, residues 39-78, purified as shown in Fig. 3B. For further explanation see text. Residue Observed Ser/Thr glycosylationa Predicted Ser/Thr glycosylation TTTR-PSM-T3 OG GTTTR-PSM-GII OG Average OG (S.D.) NetOglyc valueb h valuec   valued 1 2 3 4 5 % % % S2 31 33 35 33 (2) 0.29 0.11  0.45 S6 97 92 95 95 (2) 0.17 0.55+ 0.01+ S7 94 94 95 94 (0) 0.58+ 0.50+ 0.26+ S13 94 92 94 93 (1) 0.69+ 0.75+ 0.43+ S14 62 65 71 66 (4) 0.12 0.52+ 0.16+ S17 87 85 87 86 (1) 0.48 0.25+ 0.41+ T22 101 83 85 90 (8) 0.75+ 0.17  0.07 S23 96 88 91 92 (3) 0.84+ 0.32+  0.33 T29 75 82 79 (4) 0.32 0.78+ 0.00+ T30 79 80 83 81 (2) 0.88+ 0.83+ 0.94+ S32 96 62 56 71 (18) 0.60+ 0.47+ 0.44+ S33 97 88 79 88 (4) 0.24 0.59+ 0.65+ T37 88 79 83 83 (4) 0.88+ 0.15 0.12 T39 91 81 81 92 90 87 (5) 0.45 0.56+ 0.35+ S43 92 92 92 95 93 (1) 0.52+ 0.17 0.89+ S47 67 62 54 59 61 (5) 0.27 0.45+  0.02 T49 81 81 93 83 85 (5) 0.20 0.59+  0.18 T50 80 83 90 80 83 (4) 0.74+ 0.66+ 0.60+ T52 73 80 92 65 78 (10) 0.34 0.38+ 0.08+ S54 45 23 39 35 36 (8) 0.42 0.46+ 0.12+ S57 92 66 85 92 84 (11) 0.52+ 0.61+  0.20 S59 88 92 75 83 85 (6) 0.34 0.59+ 0.51+ T60 77 77 105 79 85 (12) 0.82+ 0.85+ 0.21+ S62 62 48 15 42 (20) 0.32 0.80+ 0.49+ S63 58 66 37 54 (12) 0.79+ 0.87+ 0.50+ S64 71 75 58 68 (7) 0.55+ 0.91+ 0.17+ S66 78 87 77 81 (4) 0.40 0.82+ 0.87+ T70 79 107 71 86 (15) 0.56+ 0.48+ 0.28+ S73 86 40 67 64 (19) 0.48 0.30+ 0.14+ T79 73 73 0.24 0.27+  0.02 S80 118 118 0.26 0.11  0.61 Average 78 a OG represents a glycosylated Ser or Thr. Numbered columns represent different sequencing experiments. b Hansen et al. (10). c Elhammer et al. (9). d Chou (11).

An examination of sequence determinations 1-5, Table I, reveals that Ser², Ser¹⁴, Ser³², Ser⁴⁷, Ser⁵⁴, Ser⁶², and Ser⁶³ are consistently the least glycosylated. As a group, 74% of the Ser residues are glycosylated compared with 83% for the Thr residues, values consistent with the ¹³C NMR data analysis (data not shown, see Ref. 26). Combined, 78% of the Ser and Thr residues are glycosylated (Table I).

The average sequence-specific glycosylation obtained from determinations 1-5 along with the O-glycosylation predictions of Hansen et al. (10) (NetOglyc activity values), Elhammer et al. (9) (h values) and Chou (11) ( values) are tabulated in Table I. For the predictions, plus symbols to the right of the value indicate that the residue is predicted to be glycosylated based on the original published cutoff criteria, and minus symbols indicate the residue would not be glycosylated. As evident from the table and visually when the observed versus predicted glycosylations are plotted together (Fig. 7), the predictions do not completely agree with each other nor do they correlate well with the observed glycosylation.

DISCUSSION

These studies demonstrate that the partial deglycosylation of mucin-type O-linked glycoproteins by mild TFMSA yields glycoprotein derivatives with monosaccharide -GalNAc side chains that are both susceptible to protease digestion and suitable for standard amino acid sequencing. Using this approach the heterogeneously glycosylated 81-residue PSM tryptic tandem repeat has been isolated and quantitatively sequenced, revealing its glycosylation pattern.

There are numerous reports of the use of automated Edman sequencing for the semiqualitative sequencing of O-linked glycoproteins and for the determination of the in vitro glycosylation patterns of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase. Sites of glycosylation have typically been estimated by the presence of "blank" cycles (see for example Refs. 23, 35-36). Other studies of the GalNAc transferase have relied on the incorporation of radiolabeled UDP-GalNAc into substrate peptide and scintillation counting of the released products after Edman sequencing (see Refs. 9, 18-19, 34, 37). Few workers have attempted to chromatographically characterize or quantitatively analyze the resultant glycosylated Ser and Thr PTH derivatives obtained from standard Edman sequencing. Abernethy and co-workers (34) have described and partially characterized the -GalNAc-O-Thr-PTH-derivative derived from the sequencing of a series of glycopeptide acceptors of the GalNAc transferase. Although they failed to demonstrate its use, these workers had suggested that a TFMSA-Edman sequencing approach would be useful for characterizing -GalNAc-Thr containing glycopeptides.³ In the laboratory of Gooley and co-workers (38-41), an Edman sequencing protocol has been developed, using the more hydrophilic solvent trifluoroacetic acid, for the sequence analysis of O-linked glycoproteins containing intact oligosaccharides.⁴ Unfortunately, for most glycoproteins with intact oligosaccharide side chains, the presence of heterogeneous oligosaccharide structures complicates the sequence analysis. Thus, quantitation of the extent of glycosylation is still a difficult task. Another drawback of this approach is that the presence of full-length oligosaccharide side chains will interfere with protease digestions making the isolation of reasonably sized glycopeptides with homogeneous peptide sequences difficult. Therefore, for several reasons, the use of mild TFMSA to trim heterogeneous O-linked oligosaccharide side chains to homogeneous peptide-linked GalNAc residues followed by standard Edman sequencing may be the most effective approach for determining the primary glycosylation pattern of heavily O-glycosylated glycoprotein domains.

The glycosylation pattern obtained for the PSM tandem repeat reveals that all potential glycosylation sites can be glycosylated, although to different extents. Interestingly, the Ser residues display a wider range of observed glycosylations, ranging from about 30% to nearly 100%, whereas the Thr residues show a much narrower range of glycosylation, ranging between 70 and 90%. The extent of Ser O-glycosylation of the PSM tandem repeat is higher than expected based on the in vitro glycosylation studies of the isolated porcine UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (14-15). With this enzyme and other GalNAc transferases isolated to date (see Refs. 9, 17-20, 35, 42-43), Ser residues are typically very poor substrates in in vitro glycosylation studies. In contrast, in vivo glycosylation studies reveal higher extents of O-glycosylation at both Ser and Thr (20). As expected the observed PSM glycosylation pattern is consistent with these observations.

As shown in Fig. 7, none of the three most recent peptide O-glycosylation predictive approaches were capable of successfully predicting the PSM tandem repeat glycosylation pattern. A comparison of the figure suggests that Elhammer's and Chou's (9, 11) predictions may be more useful for mucins as they correctly predicted the largest number of glycosylated residues. Unfortunately, none of the approaches performed well in predicting the poorly glycosylated residues, although all three approaches predicted the least glycosylated residue, Ser², to be nonglycosylated. The inability for the predictions to reasonably predict the PSM glycosylation pattern may arise from several factors, most notably mixing the glycosylation patterns of globular proteins and mucin-like domains in constructing the algorithms, the lack of accurate and complete glycosylation data, and the possible existence of several tissue-/species-specific transferases with different substrate specificities (21-23).

Having available such a large data base in the PSM tandem repeat (i.e. a total of 31 different glycosylated Ser and Thr residues), an attempt was made to determine whether any specific patterns could be associated with a given sequence's degree of glycosylation. In Tables II and III we have listed, in order of increasing extent of glycosylation, the heptad peptide sequences for each Ser and Thr residue in the tandem repeat. In addition, due to the large number of Ser/Thr dyad sequences (9 pairs) and the presence of a single Ser triad, we have also listed their peptide sequences in Table IV for comparison.⁵

Table II. PSM tandem repeat glycosylation-sequence correlations for Ser Superscripts indicate absolute relative positions (+/) to the glycosylated Ser. Serine residue Observeda OG (S.D.) Ser-OG sequence Ser/Thr dyadb +11 Gly +22 Gly +33 Gly Pro R/E I/V Ser/Thr Second strand consensusc  321 0 +1+2+3 % S2 33 (2)    SRI S VAG + +2 +1+1 +3 cee e eec S54 36 (8)    GTV S GAS + + +1 +2+3 eee e ecc S62 42 (20)    STG S SSG ++ + + +1+2+2+3 ccc c ccc S63 54 (12)    TGS S SGS ++ ++ +1+1+3+3 ccc c ccc S47 61 (5)    VAG S GTT + +3 +3+3 eec c cec S73 64 (19)    TGA S IGQ ++ +1 +3 ccc e ccc S14 66 (4)    AVS S GAS + + +2 +1+3 ccc c ccc S64 68 (7)    GSS S GSP ++ + + +3 +1+2+3 ccc c ccc S32 71 (18)    TTA S SVG + + +2 +1+2+3 ccc c eec S66 81 (4)    SSG S PGA + + +1 +2+3 ccc c ccc S57 84 (11)    SGA S GST + + +2+3+3 eec c ccc S59 85 (6)    ASG S TGS + + + +2+2+3 ccc c ccc S17 86 (1)    SGA S QAA + +3 ccc c ccc S33 88 (7)    TAS S VGV + + +1+3 +1+3 ccc e eee S23 92 (3)    AGT S GAG + + + + +1 ctc c ccc S13 93 (1)    PAV S SGA + + +3 +1 +1 ccc c ccc S43 93 (1)    ARP S VAG + +1 +2 +1 ccc e eec S7 94 (0)    AGS S GAP + + + +3 +1 ecc c ccc S6 95 (2)    VAG S SGA + + + +3 +1 eec c ccc S80d 100 ()    PET S RIS + +3 +1+2 +2 +1+3 ccc c eee Average total 74 Average %OG, +e 79 73 83 62 88   73   Average %OG, e 67 76 63 81 68   76   a Data from Table II. b ++ indicates a triad sequence. c Predicted secondary structures; c, coil; e, extended b strand; t, turn (29). d Single determination value, see Footnote 5. e Average glycosylation value of sequences containing (+) or lacking () the indicated residue(s).

Table III. PSM tandem repeat glycosylation-sequence correlations for Thr Superscripts indicate absolute relative positions (+/) to the glycosylated Thr. Threonine residue Observeda OG (S.D.) Thr-OG sequence Ser/Thr dyad +11 Gly +22 Gly +33 Gly Pro R/E V/I Ser/Thr Second strand consensusb  321 0 +1+2+3 % T79c 73 ()     QPE T SRI + +2 +2+1 +3 +2 ccc c cee T52 78 (10)     TTG T VSG + + +1 +2+2+3 cee e eee T29 79 (4)     GPG T TAS + + + +2 +1+3 ccc c ccc T30 81 (2)     PGT T ASS + + +3 +1+2+3 ccc c ccc T37 83 (4)     VGV T ETA + +1 +1+3 +2 eee e ecc T50 83 (4)     SGT T GTV + + + +3 +1+2+3 cce c eee T49 85 (5)     GSG T TGT + + + + +1+2+3 ccc e cee T60 85 (12)     SGS T GSS + + + +1+2+3+3 ccc c ccc T70 86 (15)     PGA T GAS + + +3 +3 ccc c ccc T39 87 (5)     VTE T ARP +3 +2+1 +3 +2 eee e ccc T22 90 (8)     AAG T SGA + + + +1 cct c ccc Average 83 Average %OG, +d 82 83 85 80 78   81   Average %OG, d 84 81 79 84 86   84   a Data from Table II. b Predicted secondary structures; c, coil; e, extended b strand; t, turn (29). c Single determination value, see Footnote 5. d Average glycosylation value of sequences containing (+) or lacking () the indicated residue(s).

Table IV. PSM tandem repeat dyad/triad glycosylation correlations Superscripts indicate absolute relative positions (+/) to the nearest glycosylated Ser or Thr. Residue dyads Individuala OG Minimumb OG Randomb OG Maximumb OG Sequence +11 Gly +22 Gly +33 Gly Pro R/E I/V Ser/Thr Second strand consensusc  0 +0  321 0+0 +1+2+3 % % % % T79S80d 73 100 73 73 73    QPE TS RIS +2 +1+1 +2 +3 ccc cc eee S32S33 71 88 59 61 71    TAA SS VGV + +1+3 +3 ccc ce eee S13S14 93 66 59 61 66    PAV SS GAS + +3 +1 +3 ccc cc ccc T29T30 79 81 60 64 79    GPG TT ASS + + +2 +2+3 ccc cc cce T49T50 85 83 68 71 83    GSG TT GTV ++ + +3 +2+2 ccc ec eee S59T60 85 85 70 72 85    ASG ST GSS ++ +2+2+3 ccc cc ccc T22S23 90 92 82 83 90    AAG TS GAG ++ + cct cc ccc S6S7 95 94 89 89 94    VAG SS GAP ++ +3 +3 eec cc ccc Dyad average 84 86 70 72 80  Average ++e 79  Average +/f 65 Triad  0, 0, +0  321 00+0 +3+2+3 S62SS64 42 54 68 0 15 42    SIG SSS GSP ++ +3 +2+2+3 ccc ccc ccc a Data from Table II. b Extent that each dyad (triad) may be fully glycosylated; minimum, theoretical minimum value (full negative cooperativity between sites); random, value obtained if diglycosylation is random (no cooperativity); and maximum, theoretical maximum value (full positive cooperativity between sites). c Predicted secondary structures; c, coil; e, extended b strand; t, turn (29). d Values for Thr79 and Ser80 are single determinations, see Footnote 5. e Average random glycosylation of sequences containing two Gly at the +1 and 1 positions. f Average random glycosylation of sequences containing none or one Gly at the +1 or 1 positions.

An examination of the Ser peptide data (Table II) suggests several possible trends. Sequences with Gly at positions +2 or 2 from the potential site of glycosylation appear to be associated with a higher degree of glycosylation; Gly at positions +3 or 3 may be associated with reduced glycosylation, and Gly at positions +1 or 1 apparently are glycosylation neutral as shown by their average glycosylation values at the bottom of Tables II, III, IV. Sequences containing Pro are usually more heavily glycosylated, with the Pro typically located at the +3 or 3 positions, and sequences with high Ser/Thr contents appear to be more poorly glycosylated. No obvious correlation with the extent of glycosylation beyond the uniform presence of coil and extended secondary structures is found. The presence of large hydrophobic residues, Ile and Val, does not apparently directly correlate with the extent of glycosylation (however, their positions may be important as discussed below). These trends also appear for Thr and the dyad and triad peptide sequences (Tables III and IV), although since their ranges in glycosylation are much smaller the trends are less apparent. It is noteworthy that 7 of the 9 dyad sequences, Table IV, have at least 1 Gly residue at dyad positions +1 or 1 while 4 of the sequences have Gly residues at both +1 and 1 dyad positions.⁶

To examine the possibility that the observed extent of glycosylation could be rationalized in terms of specific peptide conformation and structure, we built models for the peptide sequences in Tables II, III, IV. An extended -conformation⁷ was found to best account for the nearest neighbor and penultimate positional effects of Gly. In a -conformation, nearest neighbor residues will have their side chains directed away from each other on opposite sides of the peptide backbone, whereas penultimate residues (at positions +2 or 2) will have their side chains adjacent to each other on the same side of the peptide backbone. Thus, the observation that penultimate Gly residues may favor glycosylation suggests that penultimate residues with larger side chains may interfere with glycosylation. Furthermore, since the side chains of residues at positions +1 and 1 would not be expected to sterically interfere with the Ser or Thr side chain, the presence or absence of residues containing bulky side chains at these positions would be expected to have little effect on glycosylation. Thus, Ser², Ser⁵⁴, and Ser⁴⁷ which have N- and C-terminal penultimate residues with side chains are poorly glycosylated, whereas Ser⁶⁶, Ser⁵⁷, and Ser¹⁷ which have a single penultimate neighbor with side chains are more highly glycosylated. Ser⁷³ having 2 penultimate Gly residues might also be expected to be highly glycosylated; however, it is only moderately glycosylated. The reduced glycosylation of Ser⁷³ is consistent with the results of in vitro glycosylation studies that suggest the added flexibility of multiple Gly residues may reduce glycosylation (see Refs. 15 and 45). All of the single residue Thr sequences in Table III are highly O-glycosylated, and the ranking of Thr⁵², Thr³⁷, and Thr⁷⁰ would follow the order of decreasing penultimate residue side chain size. Ser⁴³ and Thr³⁹ appear to be exceptions to the "rule" in that they contain 2 penultimate residues with side chains and are nevertheless very highly glycosylated. We suggest that other factors, such as the presence of Pro residues in their sequences, may be responsible for enhancing their glycosylation, as discussed below.

The unexpectedly high glycosylation of Ser⁴³ may be rationalized in terms of the conformational effects of 1 Pro. Model building reveals that the Pro residue preceding Ser⁴³ can alter the peptide backbone conformation so that the 2 side chain would no longer be adjacent to the Ser side chain. The proposed conformational effects of 1 Pro may explain the elevated incidence for observing Pro at this position at known O-glycosylation sites (9-10, 16, 46). Experimentally, the introduction of a 1 Pro has been shown to enhance the in vitro glycosylation of a human von Willebrand factor dodecapeptide (