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Mass spectrometry-based analyses showing the effects of secretor and blood group status on salivary N-glycosylation.

Albertolle ME, Hassis ME, Ng CJ, Cuison S, Williams K, Prakobphol A, Dykstra AB, Hall SC, Niles RK, Ewa Witkowska H, Fisher SJ - Clin Proteomics (2015)

Bottom Line: The results revealed novel salivary N-glycosites and glycoproteins not previously reported.As compared to the secretor, nonsecretor saliva had higher levels of N-glycosylation albeit with simpler structures.Together, the results suggested a molecular basis for inter-individual variations in salivary protein glycosylation with functional implications for oral health.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143 USA ; Sandler-Moore Mass Spectrometry Core Facility, University of California San Francisco, San Francisco, CA 94143 USA.

ABSTRACT

Background: The carbohydrate portions of salivary glycoproteins play important roles, including mediating bacterial and leukocyte adhesion. Salivary glycosylation is complex. Many of its glycoproteins present ABO and Lewis blood group determinants. An individual's genetic complement and secretor status govern the expression of blood group antigens. We queried the extent to which salivary glycosylation varies according to blood group and secretor status. First, we screened submandibular/sublingual and parotid salivas collected as ductal secretions for reactivity with a panel of 16 lectins. We selected three lectins that reacted with the largest number of glycoproteins and one that recognized uncommon lactosamine-containing structures. Ductal salivas representing a secretor with complex blood group expression and a nonsecretor with a simple pattern were separated by SDS-PAGE. Gel slices were trypsin digested and the glycopeptides were individually separated on each of the four lectins. The bound fractions were de-N-glycosylated. LC-MS/MS identified the original glycosylation sites, the peptide sequences, and the parent proteins.

Results: The results revealed novel salivary N-glycosites and glycoproteins not previously reported. As compared to the secretor, nonsecretor saliva had higher levels of N-glycosylation albeit with simpler structures.

Conclusions: Together, the results suggested a molecular basis for inter-individual variations in salivary protein glycosylation with functional implications for oral health.

No MeSH data available.


Related in: MedlinePlus

Experimental workflow used to identify salivary N-glycosites. Parotid and SMSL salivas collected as the ductal secretions were separated by preparative SDS-PAGE followed by rastering of the gel bands and in-gel trypsin digestion. N-glycopeptides were captured from the peptide extracts via their ability to bind to at least one of four lectins (AAL, JAC, WGA and/or LEA). After elution and PNGase F removal of N-linked glycans, LC–MS/MS was performed to identify the original sites of oligosaccharide attachments and the protein scaffolds
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Fig2: Experimental workflow used to identify salivary N-glycosites. Parotid and SMSL salivas collected as the ductal secretions were separated by preparative SDS-PAGE followed by rastering of the gel bands and in-gel trypsin digestion. N-glycopeptides were captured from the peptide extracts via their ability to bind to at least one of four lectins (AAL, JAC, WGA and/or LEA). After elution and PNGase F removal of N-linked glycans, LC–MS/MS was performed to identify the original sites of oligosaccharide attachments and the protein scaffolds

Mentions: An overview of the general method that we devised is shown in Fig. 2. SMSL or parotid salivas were separated by preparative SDS-PAGE. The entire gel was horizontally rastered, macerated and subjected to trypsin digestion. The resulting mixture of peptides and glycopeptides was separated on an immobilized lectin column. The bound glycopeptides were eluted and treated with PNGase F, removing the N-linked oligosaccharides and converting the asparagines to aspartic acids. LC–MS/MS of the digest enabled sequencing of the peptides and identification of the original sites of carbohydrate attachment.Fig. 2


Mass spectrometry-based analyses showing the effects of secretor and blood group status on salivary N-glycosylation.

Albertolle ME, Hassis ME, Ng CJ, Cuison S, Williams K, Prakobphol A, Dykstra AB, Hall SC, Niles RK, Ewa Witkowska H, Fisher SJ - Clin Proteomics (2015)

Experimental workflow used to identify salivary N-glycosites. Parotid and SMSL salivas collected as the ductal secretions were separated by preparative SDS-PAGE followed by rastering of the gel bands and in-gel trypsin digestion. N-glycopeptides were captured from the peptide extracts via their ability to bind to at least one of four lectins (AAL, JAC, WGA and/or LEA). After elution and PNGase F removal of N-linked glycans, LC–MS/MS was performed to identify the original sites of oligosaccharide attachments and the protein scaffolds
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4696288&req=5

Fig2: Experimental workflow used to identify salivary N-glycosites. Parotid and SMSL salivas collected as the ductal secretions were separated by preparative SDS-PAGE followed by rastering of the gel bands and in-gel trypsin digestion. N-glycopeptides were captured from the peptide extracts via their ability to bind to at least one of four lectins (AAL, JAC, WGA and/or LEA). After elution and PNGase F removal of N-linked glycans, LC–MS/MS was performed to identify the original sites of oligosaccharide attachments and the protein scaffolds
Mentions: An overview of the general method that we devised is shown in Fig. 2. SMSL or parotid salivas were separated by preparative SDS-PAGE. The entire gel was horizontally rastered, macerated and subjected to trypsin digestion. The resulting mixture of peptides and glycopeptides was separated on an immobilized lectin column. The bound glycopeptides were eluted and treated with PNGase F, removing the N-linked oligosaccharides and converting the asparagines to aspartic acids. LC–MS/MS of the digest enabled sequencing of the peptides and identification of the original sites of carbohydrate attachment.Fig. 2

Bottom Line: The results revealed novel salivary N-glycosites and glycoproteins not previously reported.As compared to the secretor, nonsecretor saliva had higher levels of N-glycosylation albeit with simpler structures.Together, the results suggested a molecular basis for inter-individual variations in salivary protein glycosylation with functional implications for oral health.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143 USA ; Sandler-Moore Mass Spectrometry Core Facility, University of California San Francisco, San Francisco, CA 94143 USA.

ABSTRACT

Background: The carbohydrate portions of salivary glycoproteins play important roles, including mediating bacterial and leukocyte adhesion. Salivary glycosylation is complex. Many of its glycoproteins present ABO and Lewis blood group determinants. An individual's genetic complement and secretor status govern the expression of blood group antigens. We queried the extent to which salivary glycosylation varies according to blood group and secretor status. First, we screened submandibular/sublingual and parotid salivas collected as ductal secretions for reactivity with a panel of 16 lectins. We selected three lectins that reacted with the largest number of glycoproteins and one that recognized uncommon lactosamine-containing structures. Ductal salivas representing a secretor with complex blood group expression and a nonsecretor with a simple pattern were separated by SDS-PAGE. Gel slices were trypsin digested and the glycopeptides were individually separated on each of the four lectins. The bound fractions were de-N-glycosylated. LC-MS/MS identified the original glycosylation sites, the peptide sequences, and the parent proteins.

Results: The results revealed novel salivary N-glycosites and glycoproteins not previously reported. As compared to the secretor, nonsecretor saliva had higher levels of N-glycosylation albeit with simpler structures.

Conclusions: Together, the results suggested a molecular basis for inter-individual variations in salivary protein glycosylation with functional implications for oral health.

No MeSH data available.


Related in: MedlinePlus