Background: The discovery and development of novel biomarkers that could facilitate early diagnosis and thus prevent the progression of atherosclerosis-related diabetes mellitus (DM), cerebral infarction (CI), and cardiovascular disease (CVD) has garnered much research interest. Notably, recent reports have described a number of highly sensitive antibody markers. In this study, we aimed to identify additional antibody markers that would facilitate screening. Methods:The amplified luminescent proximity homogeneous assay (AlphaLISA) method, which incorporates glutathione-or streptavidin-donor beads and anti-human-IgG-acceptor beads, was used to evaluate serum antibody levels in serum samples. The protein array method was used for the initial screening, and peptide arrays were used to identify epitope sites. Results:The protein array identified SH3 domain-binding protein 5 (SH3BP5) as a target antigen of serum IgG antibodies in the sera of patients with atherosclerosis. We prepared recombinant glutathione S-transferase (GST)-fused SH3BP5 protein. Peptide arrays revealed that the epitope site recognized by serum antibodies is located within amino acids 161-174 of SH3BP5. AlphaLISA revealed significantly higher serum antibody levels against both the SH3BP5 protein and peptide in patients with DM, acute-phase CI, transient ischemic attack, CVD or chronic kidney disease (CKD), than in healthy donors. Furthermore, areas under the receiver operating characteristic curves of these antibodies were higher in patients with CKD and DM than in other patients. Spearman correlation analysis revealed associations between the serum antibody levels against SH3BP5 peptide and artery stenosis, hypertension, and smoking. Conclusions:The serum anti-SH3BP5 antibody marker appears to be useful for estimating the progress of atherosclerosis and may discriminate atherosclerosis associated with hypertension and/or habitual smoking.

The superoxide-generating NADPH oxidase, dormant in resting phagocytes, is activated during phagocytosis following assembly of the membrane-integrated protein cytochrome b 558 and cytosolic factors. Among the latter are the three proteins containing Src homology 3 (SH3) domains, p67 phox , p47 phox and p40 phox . While the first two factors are indispensable for the activity, p40 phox is tightly associated with p67 phox in resting cells and is suggested to have some modulatory role. Here we describe a systematic analysis of the interaction between p40 phox and p67 phox using the yeast two-hybrid system and in vitro binding assays with recombinant proteins. Both methods unequivocally showed that the minimum requirements for stable interaction are the C-terminal region of p40 phox and the region between the two SH3 domains of p67 phox . This interaction is maintained even in the presence of anionic amphiphiles used for the activation of the NADPH oxidase, raising a possibility that it mediates constitutive association of the two factors in both resting and activated cells. The C-terminal region of p40 phox responsible for the interaction contains a characteristic stretch of amino acids designated as the PC motif, that also exists in other signal-transducing proteins from yeast to human. Intensive site-directed mutagenesis to the motif in p40 phox revealed that it plays a critical role in the binding to p67 phox . Thus the PC motif appears to represent a novel module for proteinϪprotein interaction used in a variety of signaling pathways.

Transient ischemic attack (TIA) is a predictor for cerebral infarction (CI), and early diagnosis of TIA is extremely important for the prevention of CI. We set out to identify novel antibody biomarkers for TIA and CI, and detected matrix metalloproteinase 1 (MMP1), chromobox homolog 1 (CBX1), and chromobox homolog 5 (CBX5) as candidate antigens using serological identification of antigens by recombinant cDNA expression cloning (SEREX) and Western blotting to confirm the presence of serum antibodies against the antigens. Amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) revealed that serum antibody levels were significantly higher in patients with TIA or acute-phase CI (aCI) compared with healthy donors (P < 0.01). Spearman’s correlation analysis and multivariate logistic regression analysis demonstrated that levels of anti-MMP1, anti-CBX1, and anti-CBX5 antibodies were associated with age, cigarette-smoking habits, and blood pressure. Thus, serum levels of antibodies against MMP1, CBX1, and CBX5 could potentially serve as useful tools for diagnosing TIA and predicting the onset of aCI.