Site-specific Core 1 O-Glycosylation Pattern of the Porcine Submaxillary Gland Mucin Tandem Repeat
1998; Elsevier BV; Volume: 273; Issue: 41 Linguagem: Inglês
10.1074/jbc.273.41.26580
ISSN1083-351X
AutoresThomas Gerken, Cheryl L. Owens, Murali K. Pasumarthy,
Tópico(s)Proteoglycans and glycosaminoglycans research
ResumoThe sequence-specific O-linked core 1 ([R1,R2]-β-Gal(1–3)-α-GalNAc-O-Ser/Thr) glycosylation pattern has been quantitatively determined for 30 of the 31 Ser/Thr residues in the 81-residue porcine submaxillary gland mucin tandem repeat. This was achieved by Edman amino acid sequencing of the isolated tandem repeat after selective removal of non-C3-substituted, peptide-linked GalNAc residues by periodate oxidation and subsequent trimming of the remaining oligosaccharides to peptide-linked GalNAc residues by mild trifluoromethanesulfonic acid/anisole treatment. The sequencing reveals 61% (range, 12–95%) of the peptide α-N-acetylgalactosamine (GalNAc) residues to be substituted by core 1 chains, a value in agreement with the carbon-13 NMR analysis of the native mucin. Residues with the lowest C3 substitution were typically clustered in regions of sequence with the highest densities of (glycosylated) serine or threonine. This suggests that the porcine β3-Gal, core 1, transferase is sensitive to peptide sequence and/or neighboring core GalNAc glycosylation in vivo, in keeping with earlier in vitro enzymatic glycosylation studies (Granovsky, M., Blielfeldt, T., Peters, S., Paulsen, H., Meldal, M., Brockhausen, J., and Brockhausen, I. (1994)Eur. J. Biochem. 221, 1039–1046). These results demonstrate that the O-glycan structures in mucin domains are not necessarily uniformly distributed along the polypeptide core and that their lengths can be modulated by peptide sequence. The data further suggest that hydroxyamino acid spacing may contribute to the regulation of glycan length, thereby, providing a mechanism for maintaining an optimally expanded, protease resistant, mucin conformation. The sequence-specific O-linked core 1 ([R1,R2]-β-Gal(1–3)-α-GalNAc-O-Ser/Thr) glycosylation pattern has been quantitatively determined for 30 of the 31 Ser/Thr residues in the 81-residue porcine submaxillary gland mucin tandem repeat. This was achieved by Edman amino acid sequencing of the isolated tandem repeat after selective removal of non-C3-substituted, peptide-linked GalNAc residues by periodate oxidation and subsequent trimming of the remaining oligosaccharides to peptide-linked GalNAc residues by mild trifluoromethanesulfonic acid/anisole treatment. The sequencing reveals 61% (range, 12–95%) of the peptide α-N-acetylgalactosamine (GalNAc) residues to be substituted by core 1 chains, a value in agreement with the carbon-13 NMR analysis of the native mucin. Residues with the lowest C3 substitution were typically clustered in regions of sequence with the highest densities of (glycosylated) serine or threonine. This suggests that the porcine β3-Gal, core 1, transferase is sensitive to peptide sequence and/or neighboring core GalNAc glycosylation in vivo, in keeping with earlier in vitro enzymatic glycosylation studies (Granovsky, M., Blielfeldt, T., Peters, S., Paulsen, H., Meldal, M., Brockhausen, J., and Brockhausen, I. (1994)Eur. J. Biochem. 221, 1039–1046). These results demonstrate that the O-glycan structures in mucin domains are not necessarily uniformly distributed along the polypeptide core and that their lengths can be modulated by peptide sequence. The data further suggest that hydroxyamino acid spacing may contribute to the regulation of glycan length, thereby, providing a mechanism for maintaining an optimally expanded, protease resistant, mucin conformation. N-acetylgalactosamine antifreeze glycoprotein fucose galactose N-glycoloylneuraminic acid or sialic acid porcine submaxillary mucin phenylthiohydantion trifluoromethanesulfonic acid high performance liquid chromatography. TR-PSM, trypsinized, reduced, and carboxymethylated PSM composed of the tandem repeat domains TFMSA-treated TR-PSM oxidized, eliminated, and TFMSA-treated TR-PSM oxidized, reduced, and TFMSA-treated TR-PSM. Protein O-glycosylation is a common post-translational modification of a wide range of secreted and membrane-associated proteins. Many of these glycoproteins contain highlyO-glycosylated, so called mucin-like, domains that are important to their function (1Gerken T.A. Owens C.L. Pasumarthy M. Townsend R.R. Hotchkiss A.T. Techniques in Glycobiology. Marcel Dekker, New York1997: 247-269Google Scholar, 2Varki A. Glycobiology. 1993; 3: 97-130Crossref PubMed Scopus (4963) Google Scholar, 3Van Klenken B.J. Dekker J. Buller H.A. Einerhand A.W.C. Am. J. Physiol. 1995; 269: G613-G627PubMed Google Scholar, 4Jentoft N. Trends Biol. Sci. 1990; 15: 291-296Abstract Full Text PDF PubMed Scopus (622) Google Scholar). These domains contain high numbers (>30%) of Ser and Thr, which are extensivelyO-glycosylated by oligosaccharide side chains attached via α-N-acetylgalactosamine (GalNAc).1 These domains are typically 50% or more carbohydrate by weight and commonly contain tandemly repeated amino acid sequences. Little is known of the structural and dynamic effects of multiple O-glycosylation, except that these regions are resistant to proteases and possess extended conformations (4Jentoft N. Trends Biol. Sci. 1990; 15: 291-296Abstract Full Text PDF PubMed Scopus (622) Google Scholar, 5Shogren R.L. Gerken T.A Jentoft N. Biochemistry. 1989; 28: 5525-5536Crossref PubMed Scopus (217) Google Scholar, 6Shogren R. Jentoft N. Gerken T. Jamieson A. Blackwell J. Carbohydr. Res. 1987; 160: 317-327Crossref PubMed Scopus (27) Google Scholar), making them ideal structural motifs for modifying the physical-chemical and biological properties of proteins.Mucin glycoproteins are a class of high molecular weight, highlyO-glycosylated glycoproteins secreted by the epithelium, designed to protect and lubricate the cell surface from biological, chemical, and mechanical insult. Studies on the ovine and porcine submaxillary gland mucins have shown that their O-glycan side chains (which make up ∼60–70% of their masses) are responsible for their expanded solution structure and resistance to proteases, and hence are a key component governing the protection rendering properties of mucins (4Jentoft N. Trends Biol. Sci. 1990; 15: 291-296Abstract Full Text PDF PubMed Scopus (622) Google Scholar, 5Shogren R.L. Gerken T.A Jentoft N. Biochemistry. 1989; 28: 5525-5536Crossref PubMed Scopus (217) Google Scholar). Cell surface mucins and other cell surface glycoproteins containing mucin-like domains play additionally important biological roles by their involvement for example in the immune response, inflammation, cell adhesion, and tumorigenesis (1Gerken T.A. Owens C.L. Pasumarthy M. Townsend R.R. Hotchkiss A.T. Techniques in Glycobiology. Marcel Dekker, New York1997: 247-269Google Scholar, 2Varki A. Glycobiology. 1993; 3: 97-130Crossref PubMed Scopus (4963) Google Scholar, 3Van Klenken B.J. Dekker J. Buller H.A. Einerhand A.W.C. Am. J. Physiol. 1995; 269: G613-G627PubMed Google Scholar, 4Jentoft N. Trends Biol. Sci. 1990; 15: 291-296Abstract Full Text PDF PubMed Scopus (622) Google Scholar, 7Kim Y.S. Gum J. Brockhausen I. Glycoconj. J. 1996; 13: 693-707Crossref PubMed Scopus (268) Google Scholar, 8Shimizu Y. Shaw S. Nature. 1993; 366: 630-631Crossref PubMed Scopus (176) Google Scholar, 9Hilkens J. Cancer Rev. 1988; 11–12: 25-54Google Scholar, 10Apostolopoulos V. McKenzie I.F.C. Crit. Rev. Immunol. 1994; 14: 293-309Crossref PubMed Google Scholar). Mucin-like domains also serve structural roles in a number of secreted globular proteins (11Takayasu T. Suzuki S. Kametani F. Takahashi N. Shinoda T. Okuyama T. Munekata E. Biochem. Biophys. Res. Comm. 1982; 105: 1066-1071Crossref PubMed Scopus (20) Google Scholar, 12Williamson G. Belshaw N. Noel T. Ring S. Williamson M. Eur. J. Biochem. 1992; 207: 661-670Crossref PubMed Scopus (67) Google Scholar). In many cases the mucin-like domains in these glycoproteins are important to their biological function (13Zhang W. Lachmann P. Immunology. 1994; 81: 137-141PubMed Google Scholar, 14Williamson G. Belshaw N. Williamson M. Biochem. J. 1992; 282: 423-428Crossref PubMed Scopus (73) Google Scholar, 15Reddy P. Caras I. Krieger J. Biol. Chem. 1989; 264: 17329-17336Abstract Full Text PDF PubMed Google Scholar, 16Kuwano M. Seguchi T. Ono M. J. Cell Sci. 1991; 98: 131-134PubMed Google Scholar, 17Remaley A. Wong A. Schumacher U. Meng M. Brewer Jr., H. Hoeg J. J. Biol. Chem. 1993; 268: 6785-6790Abstract Full Text PDF PubMed Google Scholar, 18Bruneau N. Nganga A. Fisher E.A. Lombardo D J. Biol. Chem. 1997; 272: 27353-27361Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar).Very little is known of the site-specific glycosylation patterns of the glycosylated domains in mucins or other O-linked glycoproteins, nor how alterations in glycosylation pattern may affect properties of a glycoprotein. The absence of specificO-glycan structural information is principally due to the lack of suitable analytical methods applicable to the characterization of such heavily O-glycosylated domains. Major practical difficulties arise from the resistance of these domains to proteolytic cleavage and their inherent oligosaccharide structural heterogeneity, thereby making it difficult to obtain suitable glycopeptides for structural analysis. Although Edman sequencing of glycoproteins containing intact O-linked glycans have been reported, the inherent oligosaccharide heterogeneity presents significant problems when attempting to quantitate a glycosylation pattern of a glycoprotein (19Gooley A.A. Williams K.L. Nature. 1997; 385: 557-559Crossref PubMed Scopus (28) Google Scholar, 20Pisano A. Jardine D.R. Packer N.H. Redmond J.W. Williams K.L. Gooley A.A. Farnsworth V. Carson W. Cartier P.K. Townsend R.R. Hotchkiss A.T. Techniques in Glycobiology. Marcel Dekker, New York1997: 299-320Google Scholar). Mass spectrometric approaches have also recently been applied to the characterization of short mucin glycopeptides (21Muller S. Goletz S. Packer N. Gooley A. Lawson AM. Hanisch F-G J. Biol. Chem. 1997; 272: 24780-24793Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 22Goletz S. Leuck M. Franke P. Karsten U. Rapid Comm. Mass Spectro. 1997; 11: 1387-1398Crossref PubMed Scopus (19) Google Scholar, 23Goletz S. Thiede B. Hanlich FG Schultz M. Peter-Katalinic J. Muller S. Sietz O. 7 Karsten U. Glycobiology. 1997; 7: 881-8898Crossref PubMed Scopus (44) Google Scholar). The analysis by mass spectrometry is extremely complex and typically requires oligosaccharide trimming to the peptide GalNAc residue, via chemical or enzymatic approaches (21Muller S. Goletz S. Packer N. Gooley A. Lawson AM. Hanisch F-G J. Biol. Chem. 1997; 272: 24780-24793Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 38Iwase H. Tanaka A. Biki Y. Kokubo T. Ishii-Karakasa Kobayashi Y. Hotta K. J. Biochem. (Tokyo). 1996; 120: 393-397Crossref PubMed Scopus (50) Google Scholar). In addition, mass spectrometry approaches are not easily made quantitative and are unreliable for the quantitative determination of glycosylation patterns.Recently we reported the nearly quantitative determination of the specific α-GalNAc-Ser/Thr core O-glycosylation pattern of the 31 Ser/Thr residues in the 81-residue glycosylated tandem repeat of porcine submaxillary gland mucin (PSM) (24Gerken T.A. Owens C.L Pasumarthy M. J. Biol. Chem. 1997; 272: 9709-9719Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Our approach involved the quantitative trimming of the mucin oligosaccharide side chains to the core GalNAc residue by treatment with trifluoromethanesulfonic acid (TFMSA)/anisole at 0 °C followed by trypsinolysis and the isolation of an ensemble of tandem repeat glycopeptides. Edman amino acid sequencing was then used to obtain the glycosylation pattern by the quantitation of specific glycosylated and nonglycosylated amino acid derivatives. Practical advantages to this approach are: 1) the reduction of oligosaccharide side chain heterogeneity by the mild TFMSA/anisole treatment; 2) the increased susceptibility the partially deglycosylated (trimmed) mucin to proteases, thereby allowing the isolation of tandem repeat monomers; and 3) the ability to readily identify and quantify monosaccharide Ser/Thr-GalNAc derivatives by standard Edman amino acid sequencing protocols. Applicability of this approach has been further demonstrated by the recent determination of the core O-glycosylation pattern of the 20-residue tandem repeat of the Muc1 isolated from human milk (21Muller S. Goletz S. Packer N. Gooley A. Lawson AM. Hanisch F-G J. Biol. Chem. 1997; 272: 24780-24793Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar).A chief drawback of the use of the above approach is the contaminant loss of oligosaccharide side chain structural information, which indeed is the chief reason that the approach works so effectively. We now report on a scheme, using this basic approach, that permits the quantitation of the core 1 ([R]-β-Gal(1–3)-α-GalNAc-O-Ser/Thr) oligosaccharide structures in a peptide sequence-dependent manner. The ability to monitor the GalNAc C3 OH substitution status relies on the ability to selectively remove non-C3 substituted peptide-linked GalNAc residues after periodate oxidation, while leaving C3-substituted GalNAc residues intact (25Gerken T.A. Gupta R. Jentoft N. Biochemistry. 1992; 31: 639-648Crossref PubMed Scopus (69) Google Scholar). In this manner only those peptide-linked GalNAc residues substituted at C3 are retained for amino acid sequencing. We have confirmed the selectivity of this procedure by studies on the antifreeze glycoprotein (AFGP) from Antarctic fish, whose side chains are exclusively β-Gal(1–3)-α-GalNAc-O-Thr (26Berman E. Allerhand A. DeVries A.L. J. Biol. Chem. 1980; 255: 4407-4410Abstract Full Text PDF PubMed Google Scholar). With this modification it is now possible to further characterize theO-glycan structures of mucins and other heavilyO-glycosylated proteins in a sequence-dependent manner. Having such information will permit the characterization of the peptide specificities of the core 1, β3-galactosyltransferase (UDP-galactose:glycoprotein-N-acetylgalactosamine β3-galactosyltransferase) that catalyzes a primary step in O-glycan biosynthesis. Using this approach we have obtained the sequence specific core 1 glycosylation pattern of 30 of the 31 Ser/Thr residues in the PSM tandem repeat. This represents the first sequence-specific determination of an oligosaccharide side chain structure of any highly glycosylated mucin-type glycoprotein. Our results indicate that in vivo the β-Gal(1–3) core 1 transferase is sensitive to peptide sequence and/or neighboring core α-GalNAc glycosylation, findings that corroborate previous in vitro enzymatic studies on glycopeptide substrates (27Brockhausen I Moller G. Merz G. Adermann K. Paulsen H. Biochemistry. 1990; 29: 10206-10212Crossref PubMed Scopus (66) Google Scholar, 28Granovsky M. Blielfeldt T. Peters S. Paulsen H. Meldal M. Brockhausen J. Brockhausen I. Eur. J. Biochem. 1994; 221: 1039-1046Crossref PubMed Scopus (62) Google Scholar).The complete polypeptide structure of PSM has recently been completed (29Eckhardt A.E. Timpte C.S. DeLuca A.W. Hill R.L. J. Biol. Chem. 1997; 272: 33204-33210Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The basic molecule is composed of a very large central highlyO-glycosylated domain flanked by small Cys-rich globular domains at both the N and C termini. The glycosylated domain is composed of approximately 100 81-residue tandem repeats with the sequence as given in Fig. 1 A(30Timpte C.S. Eckhardt A.E. Abernethy J.L. Hill R.L. J. Biol. Chem. 1988; 263: 1081-1087Abstract Full Text PDF PubMed Google Scholar). Each tandem repeat contains 31 Ser/Thr O-glycosylation sites, all of which have been observed to be O-glycosylated, although to different degrees of completion (24Gerken T.A. Owens C.L Pasumarthy M. J. Biol. Chem. 1997; 272: 9709-9719Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). We have previously suggested that the observed differential glycosylation may be partially due to the steric effects of penultimate bulky side chains (24Gerken T.A. Owens C.L Pasumarthy M. J. Biol. Chem. 1997; 272: 9709-9719Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The oligosaccharide side chain structures described for PSM range from the monosaccharide α-GalNAc-O-Ser/Thr, Tn determinant, to the tetrasaccharide α-GalNAc(1–3) [α-Fuc(1–2)]-β-Gal(1–3)-α-GalNAc-O-Ser/Thr), A blood group determinant (31Carlson D. J. Biol. Chem. 1968; 243: 616-626Abstract Full Text PDF PubMed Google Scholar). Each of these mono- through tetra- saccharides are potentially glycosylated by an α-NeuNGl residue attached to the peptide-linked GalNAc at C6, thus up to eight possible oligosaccharides can be found in PSM. The complete series of possible structures is given in Fig. 1 B. The oligosaccharide side chain composition of native PSM can be readily quantitated by carbon-13 NMR spectroscopy (32Gerken T.A. Jentoft N. Biochemistry. 1987; 26: 4689-4699Crossref PubMed Scopus (37) Google Scholar). Studies on several preparations of PSM indicate that 30–40% of the PSM side chains consist of the monosaccharide α-GalNAc, while the di-, tri-, and tetrasaccharides each contribute 0–40% of the remainder of the oligosaccharides, depending on the blood group status of each individual pig (32Gerken T.A. Jentoft N. Biochemistry. 1987; 26: 4689-4699Crossref PubMed Scopus (37) Google Scholar). Forty to fifty percent of these oligosaccharides are substituted by NeuNGl, although, the distribution of NeuNGl among the different oligosaccharides is unknown (32Gerken T.A. Jentoft N. Biochemistry. 1987; 26: 4689-4699Crossref PubMed Scopus (37) Google Scholar).EXPERIMENTAL PROCEDURESMaterialsAFGP purified from the blood of Dissostichus mawsoni(fractions AFGP 1–5) (33DeVries A.L. Komatsu A.L. Feeney R.E. J. Biol. Chem. 1970; 245: 2901-2908Abstract Full Text PDF PubMed Google Scholar) was a gift of A. DeVries, University of Illinois at Urbana-Champaign, Urbana, IL. Except where noted, chemicals and enzyme reagents were obtained from Boehringer Mannheim, Sigma, or Fisher.MethodsIsolation, Reduction, Carboxymethylation, and Trypsinolysis of PSMPorcine submaxillary gland mucin was isolated as described by Shogren et al. (34Shogren R.L. Jamieson A.M. Blackwell J. Jentoft N. Biopolymers. 1986; 25: 1505-1517Crossref PubMed Scopus (48) Google Scholar). Mucin was reduced and carboxymethylated by the methods of Gupta and Jentoft (35Gupta R. Jentoft N. Biochemistry. 1989; 28: 6114-6121Crossref PubMed Scopus (21) Google Scholar) and trypsinized as described by Gerken et al. 1997 (24Gerken T.A. Owens C.L Pasumarthy M. J. Biol. Chem. 1997; 272: 9709-9719Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), yielding the PSM glycosylated domain of tandem repeats called TR-PSM. Studies were performed on two pools of A blood group-positive TR-PSM with nearly identical oligosaccharide side chain distributions; see Table IA under "Results." These pools were also utilized for our earlier studies (24Gerken T.A. Owens C.L Pasumarthy M. J. Biol. Chem. 1997; 272: 9709-9719Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar).Table ICarbon-13 NMR characterization of PSM and its partially deglycosylated derivativesA. TR-PSM pool oligosaccharide composition (mole percent)MonoDiTriTetraSum Di + Tri + TetraPool 13818232162Pool 23619242164B. Percent Ser/Thr glycosylation and GalNAc C3 substitutionTR-PSMTTR-PSMTEOTR-PSMTROTR-PSM% R-GalNAc-O-Ser/Thr74795047% C3 substituted GalNAc63NA6360Mono through tetra saccharide structures as defined in Fig. 1 B. Oligosaccharide compositions obtained from integrations of the anomeric carbon region of each pool's carbon-13 NMR spectrum in Fig. 4 A (32Gerken T.A. Jentoft N. Biochemistry. 1987; 26: 4689-4699Crossref PubMed Scopus (37) Google Scholar). Sialic acid content not included.See text and footnote 1 for abbreviations.Value obtained from the areas of the unique di- and tetrasaccharide anomeric carbon resonances (105 and 92 ppm), in Fig. 4 A, normalized to their compositions (A above) and to the Gly α-carbon resonance (44 ppm) using the amino acid compositions of 20% Gly, 21.6% Ser, and 14.4% Thr (35Gupta R. Jentoft N. Biochemistry. 1989; 28: 6114-6121Crossref PubMed Scopus (21) Google Scholar).Values obtained from the areas of the GalNAc anomeric carbons, 100 and 98 ppm, Fig. 4, B–D, normalized to the Gly α-carbon resonance (44 ppm) using the amino acid compositions of 20% Gly, 21.6% Ser, and 14.4% Thr (35Gupta R. Jentoft N. Biochemistry. 1989; 28: 6114-6121Crossref PubMed Scopus (21) Google Scholar).Calculated from the NMR derived average oligosaccharide composition in A above.NA, not applicable, all C3 substitution information lost after mild TFMSA/anisole treatment in the absence of periodate oxidation.Relative to the %R-GalNAc-O-Ser/Thr value of TTR-PSM obtained as described in note d. Open table in a new tab ChromatographyModified PSM was fractionated on a 5 × 55-cm Sephacryl S200 (Pharmacia Biotech, Uppsala, Sweden) column as described previously (24Gerken T.A. Owens C.L Pasumarthy M. J. Biol. Chem. 1997; 272: 9709-9719Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Reverse phase HPLC separations were performed as described previously (24Gerken T.A. Owens C.L Pasumarthy M. J. Biol. Chem. 1997; 272: 9709-9719Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) using modified solvent gradients.Oxidation and Elimination of TR-PSMThe selective removal of C3-unsubstituted GalNAc (oligo)saccharide side chains was performed utilizing methods modified from Gerken et al. (25Gerken T.A. Gupta R. Jentoft N. Biochemistry. 1992; 31: 639-648Crossref PubMed Scopus (69) Google Scholar). TR-PSM (10 mg/ml) was made 0.1 m HIO4, pH 4.5, and incubated at 4 °C in the dark for 5 h or overnight. Periodate was destroyed by the addition of 0.4 mNa2S2O3, 0.1 m NaI, 0.1m NaHCO3, and the elimination begun by immediately adjusting the pH to 10.5 with 1 m NaOH. After 1 h on ice the solution was dialyzed overnight against ∼2.5 mm sodium bicarbonate buffer, pH 10.5. After dialysis the remaining oxidized oligosaccharide side chains were reduced at 0 °C by the addition of 1 m NaBH4 in 1 mNa2HPO4 (1 ml/10 mg of mucin). After 1 h, NaBH4 was destroyed with dilute acetic acid to pH ∼6.5. Best results were obtained when care was taken to keep the oxidized mucin on ice until after the NaBH4 reduction (see "Results"). After exhaustive dialysis the reaction mixture was lyophilized and fractionated on Sephacryl S200. This oxidized and eliminated mucin will be called EOTR-PSM.To reduce peptide core degradation (see "Results") that apparently occurs during the overnight elevated pH elimination procedure, an alternate procedure was developed. In this procedure, after the destruction of the periodate, the mucin was immediately reduced by NaBH4 in phosphate buffer as described above. This modified mucin will be called ROTR-PSM.Partial Deglycosylation by Mild TFMSA/Anisole and Isolation of PSM Tandem Repeat GlycopeptidesSephacryl S200-fractionated, oxidized mucin was partially deglycosylated by TFMSA/anisole (3:1) at 0 °C for 4–5 h (25Gerken T.A. Gupta R. Jentoft N. Biochemistry. 1992; 31: 639-648Crossref PubMed Scopus (69) Google Scholar) and trypsinized following the procedures of Gerken et al. (24Gerken T.A. Owens C.L Pasumarthy M. J. Biol. Chem. 1997; 272: 9709-9719Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Tandem repeat glycopeptides were isolated on Sephacryl S200 chromatography and when necessary further purified by reverse phase HPLC. After this procedure TTEOTR-PSM and TTROTR-PSM tandem repeats are obtained.Biotinylation of the Tryptic Tandem Repeat GlycopeptidePurified tandem repeat glycopeptide (TTEOTR-PSM or TTROTR-PSM) (4 mg in 1.5 ml of 66 mmNaH2CO3 containing 66% Me2SO, pH 8.2) was N-terminal biotinylated by the addition of a ∼40-fold molar excess of N-hydroxysuccinimidobiotin (Pierce) (6.5 mg in 0.2 ml of Me2SO). After 6 h, a second aliquot of N-hydroxysuccinimidobiotin in Me2SO was added, and the mixture was incubated at ambient temperature overnight. The modified tandem repeat glycopeptide was separated from low molecular weight reagents on Sephracryl S200 chromatography yielding 3 mg of N-terminal biotinylated tandem repeat after lyophilization. The biotinylated tandem repeat will be called BTTEOTR-PSM or BTTROTR-PSM.Isolation of the Large C-terminal Glu-C Tandem Repeat GlycopeptideSephacryl S200-purified biotinylated tryptic tandem repeat glycopeptide (∼3 mg/ml) in 25 mm ammonium bicarbonate, pH 7.8, was digested with ∼0.4 mg of protease Glu-C overnight at 25 °C. A second addition of Glu-C, and an additional 6–8-h incubation was performed to ensure full digestion. The digestion mixture was applied to a prewashed 1-ml bed volume immobilized avidin column (Pierce) and washed with 5 ml of 50 mm ammonium bicarbonate, pH 7.8. The pass through and wash, which contain the nonbiotinylated C-terminal Glu-C glycopeptides, were collected, pooled, and lyophilized. After Sephacryl S200 chromatography the large C-terminal glycopeptide fraction (called ABTTEOTR-PSM or ABTTROTR-PSM) gave 1.5 mg of glycoprotein after lyophilization. Both glycopeptide were characterized on reverse phase HPLC.Antifreeze Glycoprotein ModificationsAFGP (12-mg aliquots) were periodate-oxidized and eliminated or oxidized and immediately reduced as described for PSM above. Partial deglycosylation of the native and oxidized AFGP by mild TFMSA/anisole was also performed as described previously for PSM (24Gerken T.A. Owens C.L Pasumarthy M. J. Biol. Chem. 1997; 272: 9709-9719Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 25Gerken T.A. Gupta R. Jentoft N. Biochemistry. 1992; 31: 639-648Crossref PubMed Scopus (69) Google Scholar). After mild TFMSA/anisole treatment, each yielded ∼8 mg of deglycosylated AFGP, which were characterized by carbon-13 NMR spectroscopy.NMR AnalysisCarbon-13 NMR spectra of native and modified mucins were obtained at 67.9 MHz with a Bruker AC-270 spectrometer using acquisition conditions previously described (32Gerken T.A. Jentoft N. Biochemistry. 1987; 26: 4689-4699Crossref PubMed Scopus (37) Google Scholar). Oligosaccharide side chain distribution was determined from integrations of the anomeric carbon region of the carbon-13 NMR spectrum of native mucin (32Gerken T.A. Jentoft N. Biochemistry. 1987; 26: 4689-4699Crossref PubMed Scopus (37) Google Scholar). GalNAc removal after oxidation-elimination was obtained by integration of the GalNAc C1 and C5 carbons and the α-carbon resonance of Gly. Percent glycosylation was obtained by normalizing the integrals to the Gly, Ser, and Thr content of PSM reported by the amino acid analysis of Gupta and Jentoft (35Gupta R. Jentoft N. Biochemistry. 1989; 28: 6114-6121Crossref PubMed Scopus (21) Google Scholar). The Ser/Thr α-carbon resonances cannot be readily used for obtaining quantitative deglycosylation data due to overlap with other peptide α-carbon resonances, although changes in their intensities can by used to confirm Ser/Thr deglycosylation.Amino Acid SequencingPulsed liquid phase Edman degradation amino acid sequencing was performed on an Applied Biosystems Procise 494 protein sequencer (Perkin-Elmer, Applied Biosystems Div., Foster City, CA) using the manufacturer's recommended pulse liquid cycles as described previously (24Gerken T.A. Owens C.L Pasumarthy M. J. Biol. Chem. 1997; 272: 9709-9719Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Amino acid phenylthiohydantion (PTH) derivatives were chromatographed on an ABI 5-μm C18 PTH column using the fast normal I gradient program and monitored by the absorbance at 269 nm. The PTH-Ser/Thr-O-GalNAc elute as two diastereotopic peaks in the chromatogram at relatively unique positions and constant area ratios. Peak areas were measured automatically, and picomoles of PTH derivative were determined after eliminating long term cycle preview and lag for each PTH derivative by a simple base line substraction approach and correcting when necessary for overlapping peaks as described previously (24Gerken T.A. Owens C.L Pasumarthy M. J. Biol. Chem. 1997; 272: 9709-9719Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). This base line substraction approach permits the quantitation of the extent of glycosylation by eliminating long range base line contributions to the peak area from adjacent cycles. Corrections for the overlap of the second eluting PTH-Thr-O-GalNAc diastereomer with PTH-Thr were made using the previously obtained area ratios for the two PTH-Thr-O-GalNAc diastereomers obtained from fully glycosylated glycopeptides (1Gerken T.A. Owens C.L. Pasumarthy M. Townsend R.R. Hotchkiss A.T. Techniques in Glycobiology. Marcel Dekker, New York1997: 247-269Google Scholar, 24Gerken T.A. Owens C.L Pasumarthy M. J. Biol. Chem. 1997; 272: 9709-9719Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The extent of glycosylation was determined by comparing the relative picomoles of nonglycosylated and glycosylated PTH derivatives after taking into account the relative recovery the individual PTH species determined from the sequencing of fully glycosylated glycopeptides of known composition (1Gerken T.A. Owens C.L. Pasumarthy M. Townsend R.R. Hotchkiss A.T. Techniques in Glycobiology. Marcel Dekker, New York1997: 247-269Google Scholar, 24Gerken T.A. Owens C.L Pasumarthy M. J. Biol. Chem. 1997; 272: 9709-9719Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Typically 1–3 nmol (10–50 μg) of glycopeptide were sequenced to ensure sequencing to at least 40–50 residues. As shown by Table I (see "Results") the reproducibility between sequencing runs is relatively good, and typica
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