Abstract
BackgroundNearly 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.MethodsSerum 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.ResultsIntense 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.ConclusionsThis 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.
Highlights
Most secreted proteins are glycosylated, and serum glycoproteins that exhibit disease-associated glycosylation changes have potential to be biomarkers
Small amounts of contaminated protein remained in IP samples, we confirmed that the contaminant did not inhibit lectin signals by spike testing using recombinant matrix metalloproteinase-3 (MMP-3)
Because we used the same antibody for Western blot analysis and antibody-overlay lectin microarray experiments, almost all lectin signals were thought to be derived from matrix metalloproteinases (MMPs)-3
Summary
Most secreted proteins are glycosylated, and serum glycoproteins that exhibit disease-associated glycosylation changes have potential to be biomarkers. To prevent RA progression, it is important to define the treatment target, such as remission or at least low disease activity; to assess disease activity using composite measures; and to adapt therapy if the target is not achieved within a particular time frame. The Disease Activity Score in 28 joints (DAS28), which combines evaluation by a rheumatologist, laboratory test results, and the patient global assessment, has commonly been used to assess disease activity [4, 5]. New indices such as the Simplified Disease Activity Index [6] and Clinical Disease Activity Index [7], which simplified the DAS28, have been developed
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