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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

Lectin capture efficiency, in terms of number of N-glycosites identified and relative abundance. Results shown are of analysis of a single secretor and a single nonsecretor. a (upper) Overall, AAL enrichment tended to yield the greatest number of N-glycosites. a (middle) With regard to N-glycosites that were common among donors, lectin performance did not depend on secretor status (a, lower) whereas more N-glycosites tended to be captured by AAL from the nonsecretor sample. b (upper) Overall, AAL demonstrated the greatest capture efficiency, enriching more N-glycosites from the parotid saliva sample of the nonsecretor (starred). b (middle) With regard to N-glycosites that were common among donors, the low level of jacalin capture of parotid sites from the secretor sample was evident (starred). b (lower) With regard to donor-unique N-glycosites, AAL capture from parotid saliva of the nonsecretor was once again most productive in terms of a number of identified N-glycosites (starred). c (upper) As to relative abundances in terms of spectral counts and overall performance, WGA and AAL capture tended to have the highest efficiently. c (middle) In terms of donor-common species, the highest number of N-glycosites tended to be found in the AAL bound fraction of the secretor parotid saliva sample. c (lower) In terms of donor-unique sites, WGA captured the highest numbers from the nonsecretor parotid saliva sample (starred)
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Fig6: Lectin capture efficiency, in terms of number of N-glycosites identified and relative abundance. Results shown are of analysis of a single secretor and a single nonsecretor. a (upper) Overall, AAL enrichment tended to yield the greatest number of N-glycosites. a (middle) With regard to N-glycosites that were common among donors, lectin performance did not depend on secretor status (a, lower) whereas more N-glycosites tended to be captured by AAL from the nonsecretor sample. b (upper) Overall, AAL demonstrated the greatest capture efficiency, enriching more N-glycosites from the parotid saliva sample of the nonsecretor (starred). b (middle) With regard to N-glycosites that were common among donors, the low level of jacalin capture of parotid sites from the secretor sample was evident (starred). b (lower) With regard to donor-unique N-glycosites, AAL capture from parotid saliva of the nonsecretor was once again most productive in terms of a number of identified N-glycosites (starred). c (upper) As to relative abundances in terms of spectral counts and overall performance, WGA and AAL capture tended to have the highest efficiently. c (middle) In terms of donor-common species, the highest number of N-glycosites tended to be found in the AAL bound fraction of the secretor parotid saliva sample. c (lower) In terms of donor-unique sites, WGA captured the highest numbers from the nonsecretor parotid saliva sample (starred)

Mentions: Parotid and SMSL salivas from a single secretor and a single nonsecretor of the glycotypes described above were studied in this series of experiments. Figure 6a illustrates the efficiency of the lectins employed in terms of the number of N-glycosites identified summed for SMSL and parotid salivas. None of the results achieved statistical significance in terms of being greater than 2 SD above or below the mean values. However, trends were observed. With regard to the total numbers, AAL enrichment tended to lead to the highest number of identifications (top panels). There was no difference in lectin performance in terms of N-glycosites that were common among donors (middle panel). A trend was observed in which a higher number of donor-unique N-glycosites tended to be captured by AAL from the nonsecretor sample (lower panel). Next, we explored these results in terms of each saliva type (Fig. 6b). Overall, AAL was the only lectin that outperformed the others, capturing significantly more N-glycosites from the parotid saliva sample of the nonsecretor (upper Panel). With regard to N-glycosites that were common among donors, the same trend was observed (middle Panel). However, we also noted the low recovery of N-glycosites from the secretor parotid sample following jacalin capture. With regard to donor-unique sites, AAL capture from the parotid sample of the nonsecretor once again led to the highest number of unique identifications (lower panel). AAL separation also led to the greatest number of unique N-glycosite identifications per sample-lectin combination (Additional file 6: Figure S5; left Panel). However, it was the secretor-SMSL-jacalin combination that resulted in the highest fraction of recovered unique glycopeptides (Additional file 6: Figure S5; right Panel).Fig. 6


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)

Lectin capture efficiency, in terms of number of N-glycosites identified and relative abundance. Results shown are of analysis of a single secretor and a single nonsecretor. a (upper) Overall, AAL enrichment tended to yield the greatest number of N-glycosites. a (middle) With regard to N-glycosites that were common among donors, lectin performance did not depend on secretor status (a, lower) whereas more N-glycosites tended to be captured by AAL from the nonsecretor sample. b (upper) Overall, AAL demonstrated the greatest capture efficiency, enriching more N-glycosites from the parotid saliva sample of the nonsecretor (starred). b (middle) With regard to N-glycosites that were common among donors, the low level of jacalin capture of parotid sites from the secretor sample was evident (starred). b (lower) With regard to donor-unique N-glycosites, AAL capture from parotid saliva of the nonsecretor was once again most productive in terms of a number of identified N-glycosites (starred). c (upper) As to relative abundances in terms of spectral counts and overall performance, WGA and AAL capture tended to have the highest efficiently. c (middle) In terms of donor-common species, the highest number of N-glycosites tended to be found in the AAL bound fraction of the secretor parotid saliva sample. c (lower) In terms of donor-unique sites, WGA captured the highest numbers from the nonsecretor parotid saliva sample (starred)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig6: Lectin capture efficiency, in terms of number of N-glycosites identified and relative abundance. Results shown are of analysis of a single secretor and a single nonsecretor. a (upper) Overall, AAL enrichment tended to yield the greatest number of N-glycosites. a (middle) With regard to N-glycosites that were common among donors, lectin performance did not depend on secretor status (a, lower) whereas more N-glycosites tended to be captured by AAL from the nonsecretor sample. b (upper) Overall, AAL demonstrated the greatest capture efficiency, enriching more N-glycosites from the parotid saliva sample of the nonsecretor (starred). b (middle) With regard to N-glycosites that were common among donors, the low level of jacalin capture of parotid sites from the secretor sample was evident (starred). b (lower) With regard to donor-unique N-glycosites, AAL capture from parotid saliva of the nonsecretor was once again most productive in terms of a number of identified N-glycosites (starred). c (upper) As to relative abundances in terms of spectral counts and overall performance, WGA and AAL capture tended to have the highest efficiently. c (middle) In terms of donor-common species, the highest number of N-glycosites tended to be found in the AAL bound fraction of the secretor parotid saliva sample. c (lower) In terms of donor-unique sites, WGA captured the highest numbers from the nonsecretor parotid saliva sample (starred)
Mentions: Parotid and SMSL salivas from a single secretor and a single nonsecretor of the glycotypes described above were studied in this series of experiments. Figure 6a illustrates the efficiency of the lectins employed in terms of the number of N-glycosites identified summed for SMSL and parotid salivas. None of the results achieved statistical significance in terms of being greater than 2 SD above or below the mean values. However, trends were observed. With regard to the total numbers, AAL enrichment tended to lead to the highest number of identifications (top panels). There was no difference in lectin performance in terms of N-glycosites that were common among donors (middle panel). A trend was observed in which a higher number of donor-unique N-glycosites tended to be captured by AAL from the nonsecretor sample (lower panel). Next, we explored these results in terms of each saliva type (Fig. 6b). Overall, AAL was the only lectin that outperformed the others, capturing significantly more N-glycosites from the parotid saliva sample of the nonsecretor (upper Panel). With regard to N-glycosites that were common among donors, the same trend was observed (middle Panel). However, we also noted the low recovery of N-glycosites from the secretor parotid sample following jacalin capture. With regard to donor-unique sites, AAL capture from the parotid sample of the nonsecretor once again led to the highest number of unique identifications (lower panel). AAL separation also led to the greatest number of unique N-glycosite identifications per sample-lectin combination (Additional file 6: Figure S5; left Panel). However, it was the secretor-SMSL-jacalin combination that resulted in the highest fraction of recovered unique glycopeptides (Additional file 6: Figure S5; right Panel).Fig. 6

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