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. 2020 Nov 1;10(65):39739-39752.
doi: 10.1039/d0ra05466f. eCollection 2020 Oct 27.

Integrated glycomics strategy for the evaluation of glycosylation alterations in salivary proteins associated with type 2 diabetes mellitus

Hanjie Yu  1 , Junhong Wang  2 , Zhen Tang  1 , Xia Li  1 , Mengqi Yin  1 , Fan Zhang  1 , Jian Shu  1 , Wentian Chen  1 , Shuang Yang  3 , Zheng Li  1
Affiliations

Integrated glycomics strategy for the evaluation of glycosylation alterations in salivary proteins associated with type 2 diabetes mellitus

Hanjie Yu et al. RSC Adv. .

Abstract

Glycosylation is involved in several biological processes, and its alterations can reflect the process of certain diseases. Type 2 diabetes mellitus (T2DM) has attained the status of a global pandemic; however, the difference in salivary protein glycosylation between healthy subjects and patients with T2DM has not been fully understood. In the present study, salivary specimens from patients with T2DM (n = 72) and healthy volunteers (HVs, n = 80) were enrolled and divided into discovery and validation cohorts. A method combining the lectin microarray and lectin blotting was employed to investigate and confirm the altered glycopatterns in salivary glycoproteins. Then, lectin-mediated affinity capture of glycoproteins and MALDI-TOF/TOF-MS were performed to obtain the precise structural information of the altered glycans. As a result, the glycopatterns recognized by 5 lectins (LEL, VVA, Jacalin, RCA120 and DSA) showed significant alteration in the saliva of T2DM patients. Notably, the glycopattern of Galβ-1,4GlcNAc (LacNAc) recognized by LEL exhibited a significant increase in T2DM patients compared to HVs in both discovery and validation cohorts. The MALDI-TOF/TOF-MS results indicated that there were 10 and 7 LacNAc-containing N/O-glycans (e.g. m/z 1647.586, 11 688.613 and 1562.470) that were identified only in T2DM patients. Besides, the relative abundance of 3 LacNAc-containing N-glycans and 10 LacNAc-containing O-glycans showed an increase in the glycopattern in T2DM patients. These results indicated that the glycopattern of LacNAc is increased in salivary glycoproteins from T2DM patients, and an increase in LacNAc-containing N/O-glycans may contribute to this alteration. Our findings provide useful information to understand the complex physiological changes in the T2DM patients.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1. The investigation of different glycopatterns of salivary glycoproteins between HVs and T2DM patients in the discovery cohort using a lectin microarray. (A) The layout of lectin microarray. A total of 37 lectins were dissolved in the recommended buffer and spotted on lectin microarray, each lectin was spotted in triplicate per block. (B) Scanned images were obtained for the analysis of salivary glycopatterns from HVs and T2DM patients. The lectins with increased NFIs in T2DM patients are marked with red boxes, and those with decreased NFIs are marked with white boxes; (C) the NFIs of 7 lectins were significantly altered in T2DM patients compared to HVs based on fold change and the Mann–Whitney test (*p < 0.05, **p < 0.01, and ***p < 0.001). The data are presented as the averaged NFI ± SD; (D) hierarchical clustering analysis of the 7 altered lectins for all samples. Glycan profiles of salivary glycoproteins from HVs and T2DM patients were clustered (average linkage, correlation similarity). The samples were listed in columns, and the lectins were listed in rows. The color and intensity of each square indicated expression levels relative to other data in the row. Red, high; green, low; black, medium; (E) the NFIs of 7 lectins from HVs and T2DM patients were subjected to principal component analysis (PCA), HV and T2DM samples are indicated by green triangles and red triangles, respectively.
Fig. 2
Fig. 2. Validation of the differential expressions of the glycopatterns in pooled saliva from HVs and T2DM patients. (A) The salivary proteins from HV and T2DM groups were separated by 10% SDS-PAGE and transferred onto a PVDF membrane. The 30 μg of Cy 5 labelled LEL, VVA and Jacalin were incubated with membranes, and the images were acquired using a STORM fluorImager respectively. The difference protein bands between HV and T2DM samples are marked with red frames. (B) The gray value of the difference protein bands was measured using the ImageJ software.
Fig. 3
Fig. 3. MALDI-TOF/TOF-MS spectra of the sialic acid-derivatized N-linked glycan from the LEL affinity glycoproteins of the saliva from HVs and T2DM patients. The salivary glycoproteins that contained LacNAc were isolated by LEL-magnetic particle conjugates, and derivatized N-glycans from HV (A) and T2DM samples (B) were released and characterized by MALDI-TOF/TOF-MS. Detailed glycan structures were annotated using the GlycoWorkbench software. Proposed structures and their m/z values were shown for each peak. The LacNAc-containing N-glycan peaks that increased or decreased in patients with T2DM are marked with red dotted lines or yellow dotted lines, respectively. (C) Number of N-glycan peaks (LacNAc-contained N-glycan peaks labeled in red) released from LEL-isolated glycoproteins from HV and T2DM samples. (D) Cross-correlation of the LacNAc-containing N-glycan peaks from HVs and T2DM patients are presented in the Venn diagram.
Fig. 4
Fig. 4. MALDI-TOF/TOF-MS spectra of the sialic acid-derivatized O-linked glycan from the LEL affinity glycoproteins of the saliva from HVs and T2DM patients. (A) Glycan spectra of HVs and (B) glycan spectra of T2DM patients; the LacNAc-containing O-glycan peaks that increased or decreased in patients with T2DM are marked with red or yellow dotted lines, respectively. (C) Number of O-glycan peaks (LacNAc-containing O-glycan peaks labeled in red) released from LEL-isolated glycoproteins from HV and T2DM samples. (D) Cross-correlation of the LacNAc-containing O-glycan peaks from HVs and T2DM patients are presented in the Venn diagram.
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