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

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Abstract
Summary: Two human monoclonal autoantibodies, B‐33 and B‐24, were generated from the B cells of a patient with scleroderma. Both monoclonal antibodies (mAbs) were composed of µ and λ chains, and recognized cytoplasmic vesicular structures by indirect immunofluorescence on Hep‐2 cell line slides, although mAb B‐24 showed an additional diffuse cytoplasmic staining pattern. By Western blot, mAb B‐24 exhibited a polyreactive‐like binding pattern, whereas mAb B‐33 failed to recognize any electroblotted Hep‐2 antigen. The polyreactive versus monospecific behaviour of mAbs B‐24 and B‐33 was further confirmed by enzyme‐linked immunosorbent assay (ELISA) with a variety of foreign and autoantigens. The N‐terminal sequence of a protein band isolated by affinity chromatography with mAb B‐33 was identical to that of cation‐independent 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 goat anti‐CI‐MPR serum, indicating the recognition by B‐24 and B‐33 of a structure 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 for the existence of a point mutation in B‐33 located in the H‐complementarity‐determining region 3 (H‐CDR3) (position 100D), which produces a non‐conservative replacement of Gly by Ser. This single replacement appears to be responsible for the dramatic change in reactivity of human mAb B‐33. The data shown here provide new evidence of the critical role played by the H‐CDR3 region in distinguishing a polyspecific from a monospecific antibody. A population study demonstrated the existence of immunoglobulin G (IgG) reactivity against CI‐MPR/IGF‐2R in serum specimens from five individuals with different pathological conditions, thus indicating that this molecule is a potential target for the human autoimmune response.