Analysis of eluted peptides from type 1 diabetes-susceptible HLA class II molecules identified novel islet protein, heparin/heparan sulfate-interacting protein.
Identification of peptides derived from pancreatic islet and presented by type 1 diabetes-susceptible MHC class II molecules has great significance to elucidate the pathogenesis of type 1 diabetes. A bulk culture of Epstein-Barr virus-transformed B-cells, which were established from a 22-year-old type 1 diabetic woman with HLA-DR4 and -DQw8, was pulsed with the homogenate of a human embryonic pancreas-derived cell line 1B2C6, and another culture was not pulsed with antigen. Peptide fractions were obtained by treatment of affinity-purified HLA-DR and -DQ molecules with 0.1% trifluoroacetic acid, and were subjected to reverse-phase high performance liquid chromatography (RP-HPLC). The RP-HPLC profiles of peptides derived from DR molecules revealed three peaks that specifically appeared after pulsing, but no such peaks were obtained from DQ molecules. From one of these three peaks, a peptide that consisted of 14 amino acids (AKSXNHTXXNQXRK, where X represents the undetermined amino acids) was identified. This peptide was derived from heparin/heparan sulfate-interacting protein (HIP). Immunostaining of pancreatic sections using antiserum for HIP peptide revealed exclusive staining of the islets. Thus, HIP was identified as an islet protein naturally processed and presented by HLA-DR4 molecules.
Nakanishi
Koji
K
Department of Endocrinology and Metabolism, Toranomon Hospital, Tokyo, Japan. koji01@toranomon.gr.jp
Komatsu
Yasuhiko
Y
Kogawa
Noriaki
N
Matsushita
Hiroshi
H
D000483:Alleles; D000596:Amino Acids / Q000737:chemistry; D001402:B-Lymphocytes / Q000166:cytology; D001779:Blood Coagulation Factors / Q000737:chemistry* / Q000502:physiology*; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology*; D005260:Female; D005802:Genes, MHC Class II; D006683:HLA-DQ Antigens / Q000737:chemistry; D006684:HLA-DR Antigens / Q000737:chemistry; D015804:HLA-DR4 Antigen / Q000737:chemistry; D006493:Heparin / Q000378:metabolism*; D006497:Heparan Sulfate / Q000737:chemistry*; D004854:Herpesvirus 4, Human / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007515:Islets of Langerhans / Q000166:cytology* / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D010179:Pancreas / Q000196:embryology* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; D013997:Time Factors; D014269:Trifluoroacetic Acid / Q000378:metabolism
D000596:Amino Acids; D001779:Blood Coagulation Factors; D006683:HLA-DQ Antigens; D006684:HLA-DR Antigens; D015804:HLA-DR4 Antigen; D006493:Heparin; D006497:Heparan Sulfate; D010455:Peptides; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; D014269:Trifluoroacetic Acid; C104946:RPL29 protein, human