Limits...
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

Distribution of N-glycosites and glycoproteins between parotid and SMSL salivas in the pilot experiment. The results are derived from analysis of a single secretor (blood type O, Lea−, Leb−, Ley−, and MECA-79 low). SMSL saliva yielded the majority of identifications. Many fewer were found in parotid saliva or both secretions
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: Distribution of N-glycosites and glycoproteins between parotid and SMSL salivas in the pilot experiment. The results are derived from analysis of a single secretor (blood type O, Lea−, Leb−, Ley−, and MECA-79 low). SMSL saliva yielded the majority of identifications. Many fewer were found in parotid saliva or both secretions

Mentions: Prior to scaling up the analysis, we performed a pilot experiment to test the proposed workflow by analyzing 100 µL of parotid and the same amount of SMSL saliva from one donor (secretor, blood type O, Lea-, Leb−, Ley−, and MECA-79 low). The electrophoretically separated samples were rastered into 12 gel slices and chromatographed on immobilized AAL. LC–MS/MS analyses, a total of 24 runs, identified 31 N-glycosites from 21 glycoproteins (Fig. 3). To our knowledge, 4 were not previously reported in saliva: IGHG2_HUMAN@176 (Ig gamma-2 chain C region), PRB3_HUMAN@66 (Basic salivary proline-rich protein 3), PSG1_HUMAN@104 and @111 (Pregnancy-specific beta-1-glycoprotein 1), the latter two within the same tryptic peptide. The PSG1 N-glycosites sites were recently reported in the N-glycoproteome of human metastatic hepatocellular carcinoma cell lines [15]. At the glycoprotein level, IGHG2_HUMAN and PRB3_HUMAN were known salivary components [16, 17]; the detection of PSG1_Human in this body fluid was novel. This was despite the fact that the reproductive age, female donor was not pregnant at the time of sample collection. Nevertheless, this finding raises the interesting possibility that this family of glycoproteins, which are produced in large amounts by the placenta and circulate at high levels in maternal blood, can be detected in saliva during pregnancy. Whether their levels could be indicative of the placental dysfunction that is associated with complications such as preeclampsia is an interesting question that could be addressed in the future. Finally, we note that future studies are necessary to validate these new glycosites since chemical deamidation of asparagine residues during sample preparation can potentially lead to false positive identification of glycosites [18, 19].Fig. 3


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)

Distribution of N-glycosites and glycoproteins between parotid and SMSL salivas in the pilot experiment. The results are derived from analysis of a single secretor (blood type O, Lea−, Leb−, Ley−, and MECA-79 low). SMSL saliva yielded the majority of identifications. Many fewer were found in parotid saliva or both secretions
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: Distribution of N-glycosites and glycoproteins between parotid and SMSL salivas in the pilot experiment. The results are derived from analysis of a single secretor (blood type O, Lea−, Leb−, Ley−, and MECA-79 low). SMSL saliva yielded the majority of identifications. Many fewer were found in parotid saliva or both secretions
Mentions: Prior to scaling up the analysis, we performed a pilot experiment to test the proposed workflow by analyzing 100 µL of parotid and the same amount of SMSL saliva from one donor (secretor, blood type O, Lea-, Leb−, Ley−, and MECA-79 low). The electrophoretically separated samples were rastered into 12 gel slices and chromatographed on immobilized AAL. LC–MS/MS analyses, a total of 24 runs, identified 31 N-glycosites from 21 glycoproteins (Fig. 3). To our knowledge, 4 were not previously reported in saliva: IGHG2_HUMAN@176 (Ig gamma-2 chain C region), PRB3_HUMAN@66 (Basic salivary proline-rich protein 3), PSG1_HUMAN@104 and @111 (Pregnancy-specific beta-1-glycoprotein 1), the latter two within the same tryptic peptide. The PSG1 N-glycosites sites were recently reported in the N-glycoproteome of human metastatic hepatocellular carcinoma cell lines [15]. At the glycoprotein level, IGHG2_HUMAN and PRB3_HUMAN were known salivary components [16, 17]; the detection of PSG1_Human in this body fluid was novel. This was despite the fact that the reproductive age, female donor was not pregnant at the time of sample collection. Nevertheless, this finding raises the interesting possibility that this family of glycoproteins, which are produced in large amounts by the placenta and circulate at high levels in maternal blood, can be detected in saliva during pregnancy. Whether their levels could be indicative of the placental dysfunction that is associated with complications such as preeclampsia is an interesting question that could be addressed in the future. Finally, we note that future studies are necessary to validate these new glycosites since chemical deamidation of asparagine residues during sample preparation can potentially lead to false positive identification of glycosites [18, 19].Fig. 3

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