Characterization of the human submandibular/sublingual saliva glycoproteome using lectin affinity chromatography coupled to multidimensional protein identification technology

Abstract

In-depth analysis of the salivary proteome is fundamental to understanding the functions of salivary proteins in the oral cavity and to reveal disease biomarkers involved in different pathophysiological conditions, with the ultimate goal of improving patient diagnosis and prognosis. Submandibular and sublingual glands contribute saliva rich in glycoproteins to the total saliva output, making them valuable sources for glycoproteomic analysis. Lectin-affinity chromatography coupled to mass spectrometry-based shotgun proteomics was used to explore the submandibular/sublingual (SM/SL) saliva glycoproteome. A total of 262 N- and O-linked glycoproteins were identified by multidimensional protein identification technology (MudPIT). Only 38 were previously described in SM and SL salivas from the human salivary N-linked glycoproteome, while 224 were unique. Further comparison analysis with SM/SL saliva of the human saliva proteome, revealed 125 glycoproteins not formerly reported in this secretion. KEGG pathway analyses demonstrated that many of these glycoproteins are involved in processes such as complement and coagulation cascades, cell communication, glycosphingolipid biosynthesis neo-lactoseries, O-glycan biosynthesis, glycan structures-biosynthesis 2, starch and sucrose metabolism, peptidoglycan biosynthesis or others pathways. In summary, lectin-affinity chromatography coupled to MudPIT mass spectrometry identified many novel glycoproteins in SM/SL saliva. These new additions to the salivary proteome may prove to be a critical step for providing reliable biomarkers in the diagnosis of a myriad of oral and systemic diseases.

Figure 1. Lectin-affinity chromatograms of SM/SL glycoproteins

Enrichment of SM/SL glycoproteins was accomplished using [A] Canavalia ensiformis (Con A), [B] Ricinus communis (RCA-I) and [C] Ulex europaeus (UEA-I) agarose columns. Chromatograms showed the unbound fraction (flow through) of non-glycosylated proteins after using the corresponding equilibration/washing buffers (0.05 M Tris-0.15 M NaCl-0.004 M CaCl2, pH 7.0 for Con A; and 0.01 M Phosphate-0.15 M NaCl pH 7.2 for RCA-I and UEA-I) for each lectin column. Glycoprotein elution profiles for the moderate-desorbed (peak #1) and strongly-bound or retarded proteins (peak #2) were detected in the elution fraction after using two-different concentrations of competitive eluents with different pHs [Con A: 0.2 M (pH 7.2) and 0.5 M (pH 2.5) methyl α-D-mannopyranoside; RCA-I: 0.1 M (pH 7.2) and 0.25 M (pH 2.5) α-lactose; and UEA-I: 0.05 M (pH 7.2) and 0.5 M (pH 2.5) α-L-fucose buffers].

Figure 1. Lectin-affinity chromatograms of SM/SL glycoproteins

Enrichment of SM/SL glycoproteins was accomplished using [A] Canavalia ensiformis (Con A), [B] Ricinus communis (RCA-I) and [C] Ulex europaeus (UEA-I) agarose columns. Chromatograms showed the unbound fraction (flow through) of non-glycosylated proteins after using the corresponding equilibration/washing buffers (0.05 M Tris-0.15 M NaCl-0.004 M CaCl2, pH 7.0 for Con A; and 0.01 M Phosphate-0.15 M NaCl pH 7.2 for RCA-I and UEA-I) for each lectin column. Glycoprotein elution profiles for the moderate-desorbed (peak #1) and strongly-bound or retarded proteins (peak #2) were detected in the elution fraction after using two-different concentrations of competitive eluents with different pHs [Con A: 0.2 M (pH 7.2) and 0.5 M (pH 2.5) methyl α-D-mannopyranoside; RCA-I: 0.1 M (pH 7.2) and 0.25 M (pH 2.5) α-lactose; and UEA-I: 0.05 M (pH 7.2) and 0.5 M (pH 2.5) α-L-fucose buffers].

Figure 2. Comparison of SM/SL glycoprotein identifications among the three lectin-affinity chromatography methods

Only 38 (14.5%) proteins overlapped among the three lectins used. 109 (41.6%) proteins were exclusively isolated by Con A chromatography, 41 (15.6%) were separated using RCA-I chromatography and 25 (9.5%) were fractionated by UEA-I chromatography.

Figure 3. Gene Ontology annotation of the lectin-affinity N- and O-linked glycoproteome and whole saliva proteome. GO annotation by molecular function

Mapping of both proteomes by molecular function indicated that the highest percentage of proteins (48%/54%; glycoproteome/proteome) was associated with binding activities and the lowest (2%/1%; glycoproteome/proteome) with antioxidant activity. GO annotation by biological process. Allocation of proteins by biological process showed that the greatest percentage of these proteins was mapped to cellular process (20%/27%; glycoproteome/proteome) and the smallest (5%/3%; glycoproteome/proteome) to multi-organism process. GO annotation by cellular component. Distribution of proteins by cellular component demonstrated that the majority were mapped to the extracellular region part (54%/29%; glycoproteome/proteome), while the minority was located in the small ribosomal subunit (1%/3%; glycoproteome/proteome).

Figure 4. Comparisons between human salivary lectin-affinity N- and O-linked glycoproteome with other salivary proteomes

A. Venn diagram showing the overlap of proteins between the human salivary lectin-affinity N- and O-linked glycoproteome and the SM and SL human salivary N-linked glycoproteome . 38 (13.5%) proteins were found in both proteomes, whereas 224 (79.4%) were unique to the lectin-affinity N- and O-linked glycoproteome and 20 (7.1%) were distinctive to the N-glycoproteome. B. Venn diagram showing the overlap of proteins between the human salivary lectin-affinity N- and O-linked glycoproteome, human salivary N-linked glycoproteomes/Glycoprofile of the human salivary proteome (whole saliva, parotid saliva, submandibular saliva and sublingual saliva) ^,,, the human salivary sialiome in whole saliva and the hexapeptide N-linked glycoproteome in whole saliva . Only 21 (3.9%) proteins overlapped among the four glycoproteomes, whereas 241 (45.4%) were unique to the human salivary lectin-affinity N- and O-linked glycoproteome, 79 (14.9%) were distinctive to the N-linked glycoproteomes/Glycoprofile of the human salivary proteome in different types of saliva, 24 (4.5%) were exclusive to the human salivary sialiome and 166 (31.3%) were only in the hexapeptide N-linked glycoproteome. C. Venn diagram showing the overlap of glycoproteins between the lectin-affinity N-and O-linked glycoproteome and the SM/SL human saliva proteome . 137 (13.1%) proteins overlapped between the two proteomes, while 125 (12%) were unique to the lectin-affinity glycoproteome and 780 (74.9%) were exclusive to the SM/SL human saliva proteome. D. Venn diagram showing the overlap of glycosylated proteins between the lectin-affinity N-and O-linked glycoproteome and the whole saliva proteome . 133 (5.5%) proteins overlapped between the two proteomes, while 129 (5.3%) were only found in the lectin-affinity N-and O-linked glycoproteome and 2,157 (89.2%) were exclusive to the whole saliva proteome.

Figure 4. Comparisons between human salivary lectin-affinity N- and O-linked glycoproteome with other salivary proteomes

A. Venn diagram showing the overlap of proteins between the human salivary lectin-affinity N- and O-linked glycoproteome and the SM and SL human salivary N-linked glycoproteome . 38 (13.5%) proteins were found in both proteomes, whereas 224 (79.4%) were unique to the lectin-affinity N- and O-linked glycoproteome and 20 (7.1%) were distinctive to the N-glycoproteome. B. Venn diagram showing the overlap of proteins between the human salivary lectin-affinity N- and O-linked glycoproteome, human salivary N-linked glycoproteomes/Glycoprofile of the human salivary proteome (whole saliva, parotid saliva, submandibular saliva and sublingual saliva) ^,,, the human salivary sialiome in whole saliva and the hexapeptide N-linked glycoproteome in whole saliva . Only 21 (3.9%) proteins overlapped among the four glycoproteomes, whereas 241 (45.4%) were unique to the human salivary lectin-affinity N- and O-linked glycoproteome, 79 (14.9%) were distinctive to the N-linked glycoproteomes/Glycoprofile of the human salivary proteome in different types of saliva, 24 (4.5%) were exclusive to the human salivary sialiome and 166 (31.3%) were only in the hexapeptide N-linked glycoproteome. C. Venn diagram showing the overlap of glycoproteins between the lectin-affinity N-and O-linked glycoproteome and the SM/SL human saliva proteome . 137 (13.1%) proteins overlapped between the two proteomes, while 125 (12%) were unique to the lectin-affinity glycoproteome and 780 (74.9%) were exclusive to the SM/SL human saliva proteome. D. Venn diagram showing the overlap of glycosylated proteins between the lectin-affinity N-and O-linked glycoproteome and the whole saliva proteome . 133 (5.5%) proteins overlapped between the two proteomes, while 129 (5.3%) were only found in the lectin-affinity N-and O-linked glycoproteome and 2,157 (89.2%) were exclusive to the whole saliva proteome.

Figure 5. Western blot analyses of SM/SL glycoproteins isolated from Unbound (UF) and Eluted (EF) fractions

Panel A: Isoform 1 of N-acetylated-alpha-linked acidic dipeptidase-like protein was detected using goat polyclonal antibody to NAALADL1 (E-18) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The observed molecular weight for this protein was around ~85 kDa. Panel B: Alpha-(1,3)-fucosyltransferase was detected with rabbit polyclonal antibody against FUT5 (Abgent, Inc., San Diego, CA). This antibody recognized a protein of ~43 kDa. Panel C: Isoform 2 of Solute carrier family 12 member 2 (NKCC1) protein was detected using 2^ndMet rabbit antibody against amino acids 209–285 of rat NKCC1 fusion protein. The molecular weight of this protein was ~160 kDa. Panel D: Isoform TGN46 of Trans-Golgi network integral membrane protein 2 was identified with rabbit polyclonal antibody to TGN46 (Abcam, Inc., Cambridge, MA). The expected molecular weight for this glycoprotein was ~57 kDa.