title: Molecular modeling of eluted peptides from DQ6 molecules (DQB1*0602 and DQB1*0604) negatively and positively associated with type 1 diabetes.
abstract: Insulin-dependent diabetes mellitus (IDDM) is positively associated with HLA-DQ8, DQ2, and DQ6 (B*0604) and negatively with DQ6 (B*0602). The mechanisms by which the DQ molecules control the development of IDDM is not known. DQ6 (B*0602) and DQ6 (B*0604) molecules share the same DQalpha chain but differ in the beta chain by six residues at positions 9, 30, 57, 70, 86, and 87. The aim of the study was to sequence the peptides eluted from both DQ6 molecules and to determine the binding motifs and construct peptides for docking them into the DQ6 peptide binding groove by molecular modeling. EBV transformed B cell line homozygous for DQ6 and hybridoma cell line secreting DQ6 specific antibody were grown in large-scale culture. The DQ6 molecules were precipitated with solid-phase bound antibodies specific for DQ6. The dissociation of peptides from MHC was done with ultrafiltration and separation of peptides by reversed-phase HPLC, using Edman degradation. A special application of Edman degradation is pool sequencing. This approach allowed us to determine common characteristics of all peptides associated with a given MHC molecule. The precipitation of DQ6 molecules and the peptide elution were done successfully. The sequencing of the peptides from DQ6 (B*0602) identified three fractions: (1) IINEPTAAAIAYGLD (Bovine HSP70), (2) IINEPTAAAIAGLDR (Human HSP70), and (3) NPRDAKACVVHGSDLK (Na+/K+ ATPase). Peptide eluted from DQ6 (B*0604) had a sequence ADLFRGTLDPVEK with sequence homology to HSP70 (residues 307-319). We were able to predict the motifs for DQ6 from the ligands eluted. We used molecular modeling as a tool to identify plausible binding motifs for peptides. Our studies show that peptide ADLFRGTLDPVEK and NPRDAKACVVHGSDLK fit well in the respective DQ6 binding grooves. These predicted motifs should then be useful for screening of autoantigens associated with diabetes and identifying the epitopes that are likely to interact with T cells.
authors.affiliation: Department of Molecular Medicine, Karolinska Institute, Stockholm, Sweden. sanjeevi.carani@molmed.ki.se
authors.forename: C B
authors.initials: CB
authors.lastname: Sanjeevi
authors.forename: T P
authors.initials: TP
authors.lastname: Lybrand
authors.forename: S
authors.initials: S
authors.lastname: Stevanovic
authors.forename: H G
authors.initials: HG
authors.lastname: Rammensee
mesh_terms: D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D003922:Diabetes Mellitus, Type 1; D006683:HLA-DQ Antigens / Q000737:chemistry*; D059866:HLA-DQ beta-Chains; D006801:Humans; D008562:Membrane Glycoproteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
chemical_list: D006683:HLA-DQ Antigens; D059866:HLA-DQ beta-Chains; C069051:HLA-DQB1 antigen; D008562:Membrane Glycoproteins; D010446:Peptide Fragments