Quantification of Cleaved β2-Microglobulin in Serum from Patients Undergoing Chronic Hemodialysis

Abstract

Background: Patients on chronic hemodialysis are prone to develop amyloid deposits of misfolded ␤ 2 -microglobulin (␤ 2 M) in osteoarticular tissues. ␤ 2 M with various deletions/truncations and chemical modifications has been found together with structurally intact ␤ 2 M in extracts of ␤ 2 M amyloid fibrils. The state of the circulating population of ␤ 2 M molecules has not been characterized previously with high-resolution methods. Methods: We used immunoaffinity-liquid chromatography-mass spectrometry analysis of serum samples to examine whether structurally modified ␤ 2 M is generated in the circulation. In addition, we developed an immunoassay for the quantification of a cleaved ␤ 2 M variant in biological fluids based on novel monoclonal antibodies and applied this assay to patient and control sera. Results: A specific alteration compatible with the generation of lysine-58 -cleaved and truncated ␤ 2 M (⌬K58-␤ 2 M) was found in the sera of many (20%-40%) dialysis patients but not in control sera or sera from patients with cerebral amyloidosis (Alzheimer disease). Applied to patient sera, specific immunoassays revealed that dialysis, as expected, significantly lowered the total ␤ 2 M concentration, but the concentrations of ⌬K58-␤ 2 M remained unchanged after dialysis. The results also show

Modified Human Beta 2‐Microglobulin (desLys58) Displays Decreased Affinity for the Heavy Chain of MHC Class I and Induces Nitric Oxide Production and Apoptosis

“…Instead, by cleavage of MHC-I-associated b2m at the cell surface [20], soluble db2m molecules can be released to the cellular microenvironment and there perform various functions. In a previous work, we used a newly developed antibody with specificity for db2m [16] and showed that db2m in fact can bind to both human and murine cells even those lacking MHC-I expression [18], suggesting that db2m may bind to the sites other than the MHC-I complexes on the cell surfaces.…”

“…HYB 332-01 (332-01), a murine monoclonal antibody specific for human db2m variant [16], and anti-human b2m monoclonal antibody 290-03 as purified IgG1 preparations (1 mg ⁄ ml) were obtained from Statens Serum Institut (Copenhagen, Denmark). Human b2m was prepared and purified from the urine of uraemic patients [23] and db2m was produced as described previously [24].…”

“…In serum of patients undergoing chronic haemodialysis, a modified form of free b2m, desLys 58 b2-microglobulin (db2m), is present [16]. db2m is formed by proteolytic cleavage of b2m mediated by C1 complement and subsequent removal of Lys 58 by carboxypeptidase Blike activity [17] as illustrated in Fig.…”

“…1. A monoclonal antibody (332-01) with binding specificity for db2m was generated recently [16,18]. By the use of this antibody, we have previously demonstrated that db2m can bind to Abstract Beta2-microglobulin (b2m) is the light chain of major histocompatibility complex class I (MHC-I) molecules, and is a prerequisite for the binding of peptides to the heavy chain and their presentation to CD8 + T cells.…”

Novel Changes in β2-Microglobulin in Dialysis Patients

“…Indeed, glycated ␤ 2 -microglobulin has been demonstrated, and shown to induce inflammation in tissue lymphocytes, along with tumor necrosis factor, interleukin-1, and interleukin-6, in the formation of amyloid (9 ). In this issue of Clinical Chemistry, Corlin et al (10 ) confirm that cleaved ␤ 2 -microglobulin occurs and demonstrate that a cleavage product of ␤ 2 -microglobulin, ⌬K58-␤ 2 -microglobulin, behaves differently from normal ␤ 2 -microglobulin during hemodialysis.…”