Identification of cation-independent mannose 6-phosphate receptor/insulin-like growth factor type-2 receptor as a novel target of autoantibodies

Two human monoclonal autoantibodies, B-33 and B-24, were generated from the B cells of a patient with scleroderma. Both monoclonal antibodies (mAbs) w ere composed of mu and lambda chains, and recognized cytoplasmic vesicular structures by indirect immunofluorescence on Hep-2 cell line slides, althou gh mAb B-24 showed an additional diffuse cytoplasmic staining pattern. By W estern blot, mAb B-24 exhibited a polyreactive-like binding pattern, wherea s mAb B-33 failed to recognize any electroblotted Hep-2 antigen. The polyre active versus monospecific behaviour of mAbs B-24 and B-33 was further conf irmed by enzyme-linked immunosorbent assay (ELISA) with a variety of foreig n and autoantigens. The N-terminal sequence of a protein band isolated by a ffinity chromatography with mAb B-33 was identical to that of cation-indepe ndent mannose 6-phosphate receptor (CI-MPR), also known as the insulin-like growth factor type-2 receptor (IGF-2R). Immunofluorescence experiments on Hep-2 cell line slides demonstrated a striking co-localization between the staining pattern exhibited by these mAbs and the pattern obtained using a g oat anti-CI-MPR serum, indicating the recognition by B-24 and B-33 of a str ucture located predominantly in late endosomes. Sequence analysis of the V- region gene segments of B-33 and B-24 showed both to be identical, except f or the existence of a point mutation in B-33 located in the H-complementari ty-determining region 3 (H-CDR3) (position 100D), which produces a non-cons ervative replacement of Gly by Ser. This single replacement: appears to be responsible for the dramatic change in reactivity of human mAb B-33. The da ta shown here provide new evidence of the critical role played by the H-CDR 3 region in distinguishing a polyspecific from a monospecific antibody. A p opulation study demonstrated the existence of immunoglobulin G (Igc) reacti vity against CI-MPR/IGF-2R in serum specimens from five individuals with di fferent pathological conditions,thus indicating that this molecule is a pot ential target for the human autoimmune response.