Roles of Glycoproteins in the Diagnosis and Differential Diagnosis of Chronic and Latent Keshan Disease

Abstract

We aimed to explore the roles of glycoproteins in the pathogenesis of chronic and latent Keshan disease (CKD and LKD), and screen the lectins as indicators of significant differences in glycoproteins of KD saliva and serum. Blood and saliva were collected from 50 CKD, 50 LKD patients and 54 normal individuals. Saliva and serum lectin microarrays and saliva and serum microarrays were used to screen and verify the differences in the levels of lectin among the three groups. In the male saliva lectin microarray, Solanum tuberosum (potato) lectin (STL) and other 9 lectins showed differences between CKD and normal; STL and other 9 lectins showed differences between LKD and normal; Aleuria aurantia lectin (AAL) and other 15 lectins showed differences between CKD and LKD. In the female saliva microarray, Griffonia (Bandeiraea) simplicifolia lectin I (GSL-I) and other 9 lectins showed differences between CKD and normal; STL and other 7 lectins showed differences between LKD and normal; Maackia amurensis lectin I (MAL-I) and Triticum vulgaris (WGA) showed difference between CKD and LKD. In the male serum lectin microarray, Psophocarpus tetragonolobus lectin I (PTL-I) and other 16 lectins showed differences between CKD and normal; Ulexeuropaeus agglutinin I (UEA-I) and other 9 lectins showed differences between LKD and normal; AAL and other 13 lectins showed differences between CKD and LKD. In the female serum lectin microarray, WGA and other 13 lectins showed differences between CKD and normal; Euonymus europaeus lectin (EEL) and other 6 lectins showed differences between LKD and normal; MAL-I and other 14 lectins showed differences between CKD and LKD. Carbohydrate chain GlcNAc and α-Gal may play crucial roles in the pathogenesis of KD. STL may be considered the diagnostic biomarker for male CKD and LKD, while WGA may be useful in distinguishing between the two stages. STL may be considered the diagnostic biomarker for female LKD.

Keywords: Keshan disease; biomarker; glycoprotein; lectin microarray; saliva; serum.

Figure 1

Male salivary glycoprotein glycosylation patterns were determined by the binding of Cy3-labeled protein to 37 different lectins using a lectin microarray. CK: CKD, LK: LKD and Health: normal control. (a) Profiles of Cy3-labeled salivary proteins from patients with CKD, LKD, and normal control individuals bound to the lectin microarrays. The lectins, which exhibited remarkable differences in the staining intensity are marked with white frames; (b) Comparisons of the lectin-binding levels between the three groups. The bars represent mean ± SD measured from three replicate spots of lectins.

Figure 2

Female salivary glycoprotein glycosylation patterns were determined by the binding of Cy3-labeled protein to 37 different lectins using a lectin microarray. CK: CKD, LK: LKD and Health: normal control. (a) Profiles of Cy3-labeled salivary proteins from patients with CKD and LKD, and normal individuals bound to the lectin microarrays. The lectin microarrays revealed lectins that exhibited significant differences, which are marked with white frames; (b) Significant differences in the lectin levels among the three groups. The bars represent mean ± SD measured from three replicate spots of lectins.

Figure 3

Male serum glycoprotein glycosylation patterns were determined by the binding of Cy3-labeled protein to 37 different lectins using a lectin microarray. CK: CKD, LK: LKD and Health: normal control. (a) Profiles of Cy3-labeled serous proteins from patients with CKD and LKD, and normal individuals bound to the lectin microarrays. The lectin microarrays revealed lectins that exhibited significant differences, which are marked with white frames; (b) Significant differences in the lectin levels among the three groups. The bars represent mean + SD of the three biological replicates from each group.

Figure 4

Female serum glycoprotein glycosylation patterns were determined by the binding of Cy3-labeled protein to 37 different lectins using a lectin microarray. CK: CKD, LK: LKD and Health: normal control. (a) Profiles of Cy3-labeled serous proteins from patients with CKD, and LKD, and normal individuals bound to the lectin microarrays. The lectin microarrays revealed lectins that exhibited significant differences, which are marked with white frames; (b) Significant differences in the lectin levels among the three groups. The bars represent mean + SD of the three biological replicates from each group.

Figure 5

Validation of the binding of 6 lectins (PSA, STL, WGA, RCA, PHA-E+L and MAL-I) to saliva glycoproteins. (a) Scanned images of Cy3-labeled lectins bound to the saliva microarrays; (b) Box plot analysis of the data for the six groups obtained from the salivary microarray. Error bars represent 95% confidence intervals for mean values. The statistical significance of differences between groups is indicated by the p-value. (c) Scatter plot analysis of the data for the six groups obtained from the saliva microarray. Lines represent mean ± SEM. CK-M: male patients with CKD, LK-M: male patients with LKD, H-M: male normal controls, CK-F: female patients with CKD, LK-F: female patients with LKD and H-F.

Figure 6

Validation of the binding of 2 lectins (STL and PSA) to serum glycoproteins. (a) Scanned images of Cy3-labeled lectins bound to the serum microarrays; (b) Box plot analysis of the data for the three groups obtained from the serum microarray. Error bars represent 95% confidence intervals for mean values. The statistical significance of differences between groups is indicated by the p-value. (c) Scatter plot analysis of the data for the six groups obtained from the saliva microarray. Lines represent mean ± SEM. CK-M: male patients with CKD, LK-M: male patients with LKD, H-M: male normal controls, CK-F: female patients with CKD, LK-F: female patients with LKD and H-F.

Figure 7

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