title: Mutational analysis of two DR alpha residues involved in dimers of HLA-DR molecules.
abstract: Crystallographic analysis of HLA-DR1 molecules reveals a "dimer of dimers" with two reciprocal salt bridges between Glu 88 and Lys 111 of the two DR alpha chains. To determine whether these amino acids are critical for Ag presentation, we generated a panel of human B cell transfectants expressing DR alpha chains with mutations at residues 88, 111, or both. The mutant DR alpha chains, paired with endogenous DR3 beta chain, form cell surface dimers that retain epitopes recognized by a panel of anti-DR3 Abs. Replacement of Glu 88 with Ala (88A) selectively eliminates the ability to activate an alloreactive (anti-DR3) T cell clone. Mutant DR molecules with Lys substituted for Glu 88 (88K) fail to activate an alloreactive, an Ag-specific, and a peptide-specific T cell line. The DR alpha 88 mutants bind an exogenously supplied DR3-specific peptide and the mutant DR molecules migrate as dimers on SDS-PAGE, implying that their defective Ag presentation is not due to an inability to bind antigenic peptides. In contrast, substitution of Lys 111 with either Ala (111A) or Glu (111E) does not abrogate Ag presentation. Further, the defect introduced by Glu 88 to Lys mutation (88K) is not overcome by compensatory Lys to Glu mutation at position 111 (111E). Taken together, these results indicate an important functional or structural role for position 88 of the DR alpha chain, but argue against a requirement for interaction between DR alpha 88 and 111 during Ag-specific T cell stimulation.
authors.affiliation: Department of Pediatrics, Children's Hospital, Philadelphia, PA 19104, USA.
authors.forename: S
authors.initials: S
authors.lastname: Goodman
authors.forename: T
authors.initials: T
authors.lastname: Sawada
authors.forename: J A
authors.initials: JA
authors.lastname: Barbosa
authors.forename: B
authors.initials: B
authors.lastname: Cole
authors.forename: R
authors.initials: R
authors.lastname: Pergolizzi
authors.forename: J
authors.initials: J
authors.lastname: Silver
authors.forename: E
authors.initials: E
authors.lastname: Mellins
authors.forename: M D
authors.initials: MD
authors.lastname: Chang
mesh_terms: D017951:Antigen Presentation; D001402:B-Lymphocytes; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D015798:HLA-DR1 Antigen / Q000737:chemistry* / Q000235:genetics*; D015803:HLA-DR3 Antigen / Q000737:chemistry*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000276:immunology; D012967:Sodium Dodecyl Sulfate; D013329:Structure-Activity Relationship; D013601:T-Lymphocytes / Q000276:immunology; D014162:Transfection
chemical_list: D015798:HLA-DR1 Antigen; D015803:HLA-DR3 Antigen; D011993:Recombinant Fusion Proteins; D012967:Sodium Dodecyl Sulfate