Alteration of matrix metalloproteinase-3 O-glycan structure as a biomarker for disease activity of rheumatoid arthritis

Abstract

Background: Nearly all secreted proteins are glycosylated, and serum glycoproteins that exhibit disease-associated glycosylation changes have potential to be biomarkers. In rheumatoid arthritis (RA), C-reactive protein (CRP), and matrix metalloproteinase-3 (MMP-3) are widely used as serologic biomarkers, but they lack sufficient specificity or precision. We performed comparative glycosylation profiling of MMP-3 using a recently developed antibody-overlay lectin microarray technology that allows semicomprehensive and quantitative analysis of specific protein glycosylation to develop an RA-specific disease activity biomarker.

Methods: Serum was taken from patients with RA (n = 24) whose disease activity was scored using composite measures, and MMP-3 was immunoprecipitated and subjected to lectin microarray analysis. A disease activity index (DAI) based on lectin signal was developed and validated using another cohort (n = 60). Synovial fluid MMP-3 in patients with RA and patients with osteoarthritis (OA) was also analyzed.

Results: Intense signals were observed on a sialic acid-binding lectin (Agrocybe cylindracea galectin [ACG]) and O-glycan-binding lectins (Jacalin, Agaricus bisporus agglutinin [ABA], and Amaranthus caudatus agglutinin [ACA]) by applying subnanogram levels of serum MMP-3. ACG, ABA, and ACA revealed differences in MMP-3 quantity, and Jacalin revealed differences in MMP-3 quality. The resultant index, ACG/Jacalin, correlated well with disease activity. Further validation using another cohort confirmed that this index correlated well with several DAIs and their components, and reflected DAI changes following RA treatment, with correlations greater than those for MMP-3 and CRP. Furthermore, MMP-3, which generated a high ACG/Jacalin score, accumulated in synovial fluid of patients with RA but not in that of patients with OA. Sialidase digestion revealed that the difference in quality was derived from O-glycan α-2,6-sialylation.

Conclusions: This is the first report of a glycoprotein biomarker using glycan change at a local lesion to assess disease activity in autoimmune diseases. Differences in the degree of serum MMP-3 α-2,6-sialylation may be a useful index for estimating disease activity.

Keywords: Biomarker; Glycoprotein; MMP-3; Rheumatoid arthritis.

Fig. 1

Schematic overview of the antibody-overlay lectin microarray. A total of 45 kinds of lectins are spotted in triplicate on the array slide. Immunoprecipitation samples were applied to them, and glycoproteins bound to the lectins through their glycans. After washing, the lectins that bound to matrix metalloproteinase (MMP)-3 glycans were detected using anti-MMP-3-specific antibody

Fig. 2

The Agrocybe cylindracea galectin (ACG)/Jacalin index reflects rheumatoid arthritis (RA) activity. a Correlation of matrix metalloproteinase-3 (MMP-3) quantities before and after immunoprecipitation (IP). Serum MMP-3 measured by turbidimetric immunoassay and MMP-3 in IP samples measured by Western blot (WB) analysis (n = 24). b Representative data derived from antibody-overlay lectin microarray. Data are from a patient with RA who had high disease activity and a patient with RA who was in remission. Net intensity from triplicate experiments is presented as the mean ± SD. c Correlation between immunoprecipitated MMP-3 quantity and lectin signal. Patients with more than moderate disease activity are represented by open symbols; otherwise, data are shown as filled symbols. d Correlation between the ACG/Jacalin index and Disease Activity Score in 28 joints with erythrocyte sedimentation rate (DAS28-ESR). MAL_I Maackia amurensis leukoagglutinin I, SNA Sambucus nigra agglutinin, PHA(E) Phaseolus vulgaris erythroagglutinin, NPA Narcissus pseudonarcissus agglutinin, BPL Bauhinia purpurea lectin, ABA Agaricus bisporus agglutinin, LEL Lycopersicon esculentum lectin, STL Solanum tuberosum lectin, PNA peanut agglutinin, ACA Amaranthus caudatus agglutinin, MPA Maclura pomifera agglutinin, MAH Maackia amurensis hemagglutinin, WGA wheat germ agglutinin

Fig. 3

The Agrocybe cylindracea galectin (ACG)/Jacalin index is similar between males and females. Correlations between disease activity indices and (a) ACG/Jacalin and (b) serum matrix metalloproteinase-3 (MMP-3). Data from female and male patients are shown in filled and open symbols, respectively. DAS28-ESR Disease Activity Score in 28 joints with erythrocyte sedimentation rate

Fig. 4

Matrix metalloproteinase-3 (MMP-3) with elevated Agrocybe cylindracea galectin (ACG)/Jacalin signals produced in local lesions reflects the degree of sialylation. a Synovial fluid MMP-3 was measured by Western blot analysis after immunoprecipitation. Patients with RA (lanes 1–3) and patients with OA (lanes 4–6). b Representative lectin signals derived from synovial MMP-3 (mean ± SD). c The structure of T and sialyl-T antigen. Jacalin recognizes the former. d Jacalin and ACG signals compared before and after sialic acid digestion (mean ± SD). e The estimated basic structure of MMP-3 O-glycan. ABA Agaricus bisporus agglutinin, UDA Urtica dioica agglutinin, ACA Amaranthus caudatus agglutinin, WGA wheat germ agglutinin