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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

Selection of lectins for capturing human salivary N-glycosites. a The electrophoretic banding patterns of submandibular/sublingual (SMSL) and parotid (PS) salivary proteins and glycoproteins as visualized by Coomassie blue or Alcian blue silver staining, respectively. Replicate blots were screened against a panel of 16 lectins (Additional file 1: Figure S1), 4 of which are shown in this figure (b, c). The patterns of reactive bands for three lectins, which in the aggregate, reacted with salivary components over a broad molecular weight range (AAL, JAC and WGA) are shown. LEA was selected due to its reactivity with extended lactosamine units, potential sites of terminal saccharide modifications related to blood group status
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Fig1: Selection of lectins for capturing human salivary N-glycosites. a The electrophoretic banding patterns of submandibular/sublingual (SMSL) and parotid (PS) salivary proteins and glycoproteins as visualized by Coomassie blue or Alcian blue silver staining, respectively. Replicate blots were screened against a panel of 16 lectins (Additional file 1: Figure S1), 4 of which are shown in this figure (b, c). The patterns of reactive bands for three lectins, which in the aggregate, reacted with salivary components over a broad molecular weight range (AAL, JAC and WGA) are shown. LEA was selected due to its reactivity with extended lactosamine units, potential sites of terminal saccharide modifications related to blood group status

Mentions: As a first step, we screened SMSL and parotid saliva samples collected as the ductal secretions from twenty individuals for reactivity with a 16-lectin panel (Additional file 1: Figure S1). The donors were chosen as representing the spectrum of glycosylation commonly observed in the general US population with regard to the addition of carbohydrate blood group determinants to the core oligosaccharide structures of glycoproteins. We also evaluated relative expression of L-selectin carbohydrate ligands, e.g., MECA-79 reactivity, which are added to salivary components [14]. From the original twenty individuals, we selected four with the following characteristics: donor (1) secretor with blood type O, Lea+, Leb+, Ley+, low MECA-79; donor (2) secretor with blood type B, Lea+, Leb+, Ley+, low MECA-79; donor (3) secretor with blood type O, Lea+, Leb+, Ley+, low MECA-79; and donor (4) nonsecretor with blood type A, Lea–, Leb–, Ley–, high MECA-79. The 16 lectins were chosen based on their carbohydrate specificity, which spanned a wide spectrum from elements that are commonly found in the majority of N-linked structures to unusual sugar sequences and/or linkages (Table 1). The results are shown in Fig. 1. Panel A shows the electrophoretic banding patterns of SMSL and parotid, proteins (Coomassie blue staining) and glycoproteins (Alcian blue silver staining), respectively. Of the lectins that were screened, AAL, jacalin (JAC) and wheat germ agglutinin (WGA) reacted with the largest number of bands spanning the greatest molecular weight range (compare Fig. 1b with Additional file 1: Figure S1). Thus, using the rationale that they would capture the highest number of N-linked glycopeptides, they were chosen for the separation experiments. In this regard, we also included the Lycopersicon esculentum agglutinin (LEA) lectin, which has a much narrower specificity consistent with the banding pattern shown in Fig. 1c, but includes lactosamine units that could be substrates for the addition of the Le antigens.Table 1


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)

Selection of lectins for capturing human salivary N-glycosites. a The electrophoretic banding patterns of submandibular/sublingual (SMSL) and parotid (PS) salivary proteins and glycoproteins as visualized by Coomassie blue or Alcian blue silver staining, respectively. Replicate blots were screened against a panel of 16 lectins (Additional file 1: Figure S1), 4 of which are shown in this figure (b, c). The patterns of reactive bands for three lectins, which in the aggregate, reacted with salivary components over a broad molecular weight range (AAL, JAC and WGA) are shown. LEA was selected due to its reactivity with extended lactosamine units, potential sites of terminal saccharide modifications related to blood group status
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Selection of lectins for capturing human salivary N-glycosites. a The electrophoretic banding patterns of submandibular/sublingual (SMSL) and parotid (PS) salivary proteins and glycoproteins as visualized by Coomassie blue or Alcian blue silver staining, respectively. Replicate blots were screened against a panel of 16 lectins (Additional file 1: Figure S1), 4 of which are shown in this figure (b, c). The patterns of reactive bands for three lectins, which in the aggregate, reacted with salivary components over a broad molecular weight range (AAL, JAC and WGA) are shown. LEA was selected due to its reactivity with extended lactosamine units, potential sites of terminal saccharide modifications related to blood group status
Mentions: As a first step, we screened SMSL and parotid saliva samples collected as the ductal secretions from twenty individuals for reactivity with a 16-lectin panel (Additional file 1: Figure S1). The donors were chosen as representing the spectrum of glycosylation commonly observed in the general US population with regard to the addition of carbohydrate blood group determinants to the core oligosaccharide structures of glycoproteins. We also evaluated relative expression of L-selectin carbohydrate ligands, e.g., MECA-79 reactivity, which are added to salivary components [14]. From the original twenty individuals, we selected four with the following characteristics: donor (1) secretor with blood type O, Lea+, Leb+, Ley+, low MECA-79; donor (2) secretor with blood type B, Lea+, Leb+, Ley+, low MECA-79; donor (3) secretor with blood type O, Lea+, Leb+, Ley+, low MECA-79; and donor (4) nonsecretor with blood type A, Lea–, Leb–, Ley–, high MECA-79. The 16 lectins were chosen based on their carbohydrate specificity, which spanned a wide spectrum from elements that are commonly found in the majority of N-linked structures to unusual sugar sequences and/or linkages (Table 1). The results are shown in Fig. 1. Panel A shows the electrophoretic banding patterns of SMSL and parotid, proteins (Coomassie blue staining) and glycoproteins (Alcian blue silver staining), respectively. Of the lectins that were screened, AAL, jacalin (JAC) and wheat germ agglutinin (WGA) reacted with the largest number of bands spanning the greatest molecular weight range (compare Fig. 1b with Additional file 1: Figure S1). Thus, using the rationale that they would capture the highest number of N-linked glycopeptides, they were chosen for the separation experiments. In this regard, we also included the Lycopersicon esculentum agglutinin (LEA) lectin, which has a much narrower specificity consistent with the banding pattern shown in Fig. 1c, but includes lactosamine units that could be substrates for the addition of the Le antigens.Table 1

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