Roles of glycoprotein glycosylation in the pathogenesis of an endemic osteoarthritis, Kashin–Beck disease, and effectiveness evaluation of sodium hyaluronate treatment

Abstract

Background/aim: We aimed to explore the roles of glycoprotein glycosylation in the pathogenesis of Kashin–Beck disease (KBD), and evaluated the effectiveness of sodium hyaluronate treatment.

Materials and methods: Blood and saliva were collected from KBD patients before and after the injection of sodium hyaluronate. Normal healthy subjects were included as controls. Saliva and serum lectin microarrays and saliva and serum microarray verifications were used to screen and confirm the differences in lectin levels among the three groups.

Results: In saliva lectin microarray, bindings to Sophora japonica agglutinin (SJA), Griffonia (Bandeiraea) simplicifolia lectin I (GSL-I), Euonymus europaeus lectin (EEL), Maackia amurensis lectin II (MAL-II), Sambucus nigra lectin (SNA), Hippeastrum hybrid lectin (HHL), and Aleuria aurantia lectin (AAL) were higher in the untreated KBD patients than in the control group. Increased levels of HHL, MAL-II, and GSL-I in the untreated KBD patients discriminated them in particular from the treated ones. Jacalin was lower in the untreated KBD patients compared to the treated KBD and control groups. In serum lectin microarray, HHL and peanut agglutinin (PNA) were increased in the untreated KBD group in comparison to the control one. AAL, Phaseolus vulgaris agglutinin (E+L) (PHA-E+L), and Psophocarpus tetragonolobus lectin I (PTL-I) were lower in the untreated KBD patients compared to the treated KBD and control groups. Hyaluronate treatment appeared to normalize SNA, AAL, and MAL-II levels in saliva, and HHL, PNA, AAL, PTL-I, and PHA-E+L levels in serum. Saliva reversed microarray verification confirmed significant differences between the groups in SNA and Jacalin, in particular for GSL-I levels, while serum reversed microarray verification indicated that HHL, PNA, and AAL levels returned to normal levels after the hyaluronate treatment. Lectin blot confirmed significant differences in HHL, AAL, and Jacalin in saliva, and HHL, PNA, PHA-E+L, and AAL in serum.

Conclusion: HHL in saliva and serum may be a valuable diagnostic biomarker of KBD, and it may be used as follow-up for the hyaluronate treatment.

Keywords: carbohydrate chain; lectin microarray; saliva; serum; Kashin–Beck disease.

Figure 1

Layout of 37 lectins, the negative control (bovine serum albumin, BSA), and a positive marker contained in saliva or serum lectin microarray.

Figure 2

Salivary glycoprotein glycosylation patterns determined with Cy3-labeled lectins spotted onto a lectin microarray. UK: untreated KBD patients; TK: treated KBD patients; Healthy: normal controls. a. Profiles of Cy3-labeled salivary proteins from the UK, the TK, and the healthy individuals bound to the lectin microarrays. The lectin microarrays revealed significant lectins marked with white rectangles. b. Significant differences in the lectin levels between the three groups. The bars show mean ± SD of three biological replicates from each group.

Figure 3

a. Serous glycoprotein expression levels were determined with Cy3-labeled lectins using a lectin microarray. UK: untreated patients with KBD; TK: treated patients with KBD; Healthy: normal controls. The lectin microarrays revealed significant lectins marked with white rectangles. b. Significant differences in the lectin levels between the three groups. The bars show mean ± SD of three biological replicates from each group.

Figure 4

Verification of the differences in the saliva levels of the three lectins GSL-1, SNA, and Jacalin. UK: untreated patients with KBD; TK: treated patients with KBD; Healthy: normal controls. a. Scanned pictures of Cy3-labeled lectins bound to the salivary protein microarrays. b. Box plot analysis of the salivary microarray data obtained from the three groups. Error bars show 95% confidence intervals for mean values. The P-values indicate the statistical significance of differences between the groups. c. Scatter plot analysis of the salivary protein microarray data obtained from the three groups. Lines show mean ± SEM.

Figure 5

Verification of the differences in the sera levels of the three lectins HHL, PNA, and AAL. UK: untreated patients with KBD; TK: treated patients with KBD; Healthy: normal controls. a. Scanned pictures of Cy3-labeled lectins bound to the serous protein microarrays. b. Box plot analysis of the serous microarray data obtained from the three groups. Error bars show 95% confidence intervals for mean values. The P-values indicate the statistical significance of differences between groups. c. Scatter plot analysis of the serous protein microarray data obtained from the three groups. Lines show mean ± SEM.

Figure 6

Lectin blot analysis of the differential expressions of the glycopatterns in the saliva from three groups: Healthy: normal control; UK: untreated KBD; TK: treated KBD. The three groups of saliva pooled samples using 3 lectins (HHL, AAL, and Jacalin). The apparent bands belong to different molecular weights ranging from 15 to 250 kDa, which are marked as L1–L8, respectively.

Figure 7

Lectin blot analysis of the differential expressions of the glycopatterns in the sera from three groups: Healthy: normal control; UK: untreated KBD; TK: treated KBD. The three groups of saliva pooled samples using 4 lectins (HHL, PNA, PHA-E+L, and AAL). The apparent bands belong to different molecular weights ranging from 15 to 250 kDa, which are marked as L1–L10, respectively.