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) were composed of mu and lambda 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.
Tarrago
D
D
Servicio de Immunologia, Hospital Universitario Virgen del Rocio, Sevilla, Spain.
Aguilera
I
I
Melero
J
J
Wichmann
I
I
Nuñez-Roldan
A
A
Sanchez
B
B
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics*; D018771:Arthralgia / Q000276:immunology; D001323:Autoantibodies / Q000097:blood; D001324:Autoantigens / Q000097:blood*; D001483:Base Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D006801:Humans; D006825:Hybridomas; D007970:Leukopenia / Q000276:immunology; D008107:Liver Diseases / Q000276:immunology; D008360:Mannosephosphates; D008969:Molecular Sequence Data; D017354:Point Mutation; D011693:Purpura / Q000276:immunology; D011928:Raynaud Disease / Q000276:immunology; D017527:Receptor, IGF Type 2 / Q000276:immunology*; D012595:Scleroderma, Systemic / Q000276:immunology*; D017386:Sequence Homology, Amino Acid
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D001324:Autoantigens; D008360:Mannosephosphates; D017527:Receptor, IGF Type 2; C027693:mannose-6-phosphate