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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

Glycan profiles of nonsecretors vs. secretors for parotid (a and b) and SMSL (c and d) salivas. N-linked oligosaccharides were released by PNGase F digestion and analyzed by MALDI MS. Putative structures of several glycans that were detected are shown. Overall, both salivas contained high mannose and hybrid bi- and tri-antennary structures. Higher levels of fucosylation, consistent with the presence of blood group H-antigen structure (marked with black stars), were observed in the secretor samples. SMSL glycans had a higher level of fucosylation as compared to those observed in parotid saliva. Monoisotopic masses of detected glycans and their structure assignments are listed in Additional file 7: Table S2. Monosaccharides are indicated using symbols defined by the Consortium for Functional Glycomics [61]
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Fig8: Glycan profiles of nonsecretors vs. secretors for parotid (a and b) and SMSL (c and d) salivas. N-linked oligosaccharides were released by PNGase F digestion and analyzed by MALDI MS. Putative structures of several glycans that were detected are shown. Overall, both salivas contained high mannose and hybrid bi- and tri-antennary structures. Higher levels of fucosylation, consistent with the presence of blood group H-antigen structure (marked with black stars), were observed in the secretor samples. SMSL glycans had a higher level of fucosylation as compared to those observed in parotid saliva. Monoisotopic masses of detected glycans and their structure assignments are listed in Additional file 7: Table S2. Monosaccharides are indicated using symbols defined by the Consortium for Functional Glycomics [61]

Mentions: Samples from a single secretor and a single nonsecretor were analyzed. Based on donor blood group status, we theorized that the glycan profiles of the nonsecretor samples were characterized by saccharides with fewer fucosylated species. MS-based analyses confirmed this result. The parotid profiles are shown in Fig. 8a, b (nonsecretor and secretor, respectively); the SMSL profiles are shown in Fig. 8c, d (nonsecretor and secretor, respectively). Parotid saliva contained high mannose as well as hybrid, bi- and tri-antennary structures with higher levels of fucosylation in the secretor as compared to the nonsecretor sample (marked with black stars). In general, the structures we observed (Additional file 7: Table S2) were highly correlated with the core fucosylated oligosaccharides that were described by Guile et al. in their analysis of the human parotid gland glycome [30]. The same general pattern was observed in the SMSL sample with even higher levels of fucosylation. Thus, our structural analyses confirmed that, in saliva, the blood group expression patterns are borne out at the oligosaccharide structural level. We note that other groups have reported differential glycan complexity of secretors compared to nonsecretors [31, 32]. Thomsson et al. detected ABO(H) blood group specific structures that decorated O-glycans of salivary MUC5B obtained from nonsecretors demonstrating a higher degree of sialylation compared to secretors [31]. Similarly, an absence of glycan masses corresponding to H-antigen structures in saliva collected from a nonsecretor blood group A individual was observed by Everest-Dass et al. [32].Fig. 8


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)

Glycan profiles of nonsecretors vs. secretors for parotid (a and b) and SMSL (c and d) salivas. N-linked oligosaccharides were released by PNGase F digestion and analyzed by MALDI MS. Putative structures of several glycans that were detected are shown. Overall, both salivas contained high mannose and hybrid bi- and tri-antennary structures. Higher levels of fucosylation, consistent with the presence of blood group H-antigen structure (marked with black stars), were observed in the secretor samples. SMSL glycans had a higher level of fucosylation as compared to those observed in parotid saliva. Monoisotopic masses of detected glycans and their structure assignments are listed in Additional file 7: Table S2. Monosaccharides are indicated using symbols defined by the Consortium for Functional Glycomics [61]
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig8: Glycan profiles of nonsecretors vs. secretors for parotid (a and b) and SMSL (c and d) salivas. N-linked oligosaccharides were released by PNGase F digestion and analyzed by MALDI MS. Putative structures of several glycans that were detected are shown. Overall, both salivas contained high mannose and hybrid bi- and tri-antennary structures. Higher levels of fucosylation, consistent with the presence of blood group H-antigen structure (marked with black stars), were observed in the secretor samples. SMSL glycans had a higher level of fucosylation as compared to those observed in parotid saliva. Monoisotopic masses of detected glycans and their structure assignments are listed in Additional file 7: Table S2. Monosaccharides are indicated using symbols defined by the Consortium for Functional Glycomics [61]
Mentions: Samples from a single secretor and a single nonsecretor were analyzed. Based on donor blood group status, we theorized that the glycan profiles of the nonsecretor samples were characterized by saccharides with fewer fucosylated species. MS-based analyses confirmed this result. The parotid profiles are shown in Fig. 8a, b (nonsecretor and secretor, respectively); the SMSL profiles are shown in Fig. 8c, d (nonsecretor and secretor, respectively). Parotid saliva contained high mannose as well as hybrid, bi- and tri-antennary structures with higher levels of fucosylation in the secretor as compared to the nonsecretor sample (marked with black stars). In general, the structures we observed (Additional file 7: Table S2) were highly correlated with the core fucosylated oligosaccharides that were described by Guile et al. in their analysis of the human parotid gland glycome [30]. The same general pattern was observed in the SMSL sample with even higher levels of fucosylation. Thus, our structural analyses confirmed that, in saliva, the blood group expression patterns are borne out at the oligosaccharide structural level. We note that other groups have reported differential glycan complexity of secretors compared to nonsecretors [31, 32]. Thomsson et al. detected ABO(H) blood group specific structures that decorated O-glycans of salivary MUC5B obtained from nonsecretors demonstrating a higher degree of sialylation compared to secretors [31]. Similarly, an absence of glycan masses corresponding to H-antigen structures in saliva collected from a nonsecretor blood group A individual was observed by Everest-Dass et al. [32].Fig. 8

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