Molecular interactions between transfected human TCR, immunodominant myelin basic protein peptide 152-165, and HLA-DR13.

Chimeric TCR transfectants expressing human extracellular sequences and murine intracellular/transmembrane sequences were generated to analyze the trimolecular interaction between myelin basic protein (MBP) autoantigen, HLA, and a TCR isolated from a patient with multiple sclerosis. Chimeric transfectants responded to TCR activation by CD3- and TCRBV22S1-specific mAbs and by superantigen. Additionally, chimeric transfectants responded to autoantigen-specific activation with MBP 152-165 when presented by DR(alpha,beta1*1301) independent of the CD4 adhesion molecule. Transfectants did not respond to Ag presented by other HLA-DR molecules, including the closely related DR(alpha,beta1*1302). In peptide-binding studies with a panel of serial alanine-substituted MBP peptides, HLA contact residues necessary for anchoring MBP 152-165 to DR(alpha,beta1*1301) were also defined: 154 (F), 159 (R), and 162 (R). The chimeric TCR transfectant's differential response to a similar panel of MBP analogues defined residues that interact with the TCR: 153 (I), 155 (K), 156 (L), 160 (D), and 161 (S). Analysis of molecular interactions, such as those described in this work, may be central to developing new strategies for suppressing Ag-specific responses in human autoimmune disease.

The relative importance of individual DR binding motif positions as defined by peptide anchor analysis of influenza hemagglutinin peptide 306-318 and human myelin basic protein peptide 152-165 binding to several DR molecules: definition of a common extended DR binding motif.

Definition of peptide binding motifs for DR molecules has proven difficult as the peptides that bind to a DR molecule have shown extensive variability at putative motif positions. Recent studies suggest that specific peptide anchor residues (motif positions) and specific DR residues can differ in importance for peptide binding to a DR molecule. To assess further the relevance of individual peptide anchor residues, the binding of serial alanine-substituted analogs of influenza virus hemagglutinin (HA) 306-318 and human myelin basic protein (MBP) 152-165 to a panel of transfected wild-type DR molecules was examined. This analysis included DR molecules from a wide range of allelic families and, unlike most earlier studies, multiple members of single DR allelic families. The data show that different peptide residues serve as critical anchors for binding to different DR molecules. For example, MBP binding to DR(alpha, beta 1*0303) required peptide residues F154 (i), R159 (i + 5) and R162 (i + 8). In contrast, MBP binding to DR(alpha, beta 1*0102) required peptide residues I153 (i) and L156 (i + 3). More importantly, the combination of critical anchor residues in HA and MBP differed for binding to a single DR molecule [e.g. V309 (i) for HA and I153 (i) and L156 (i + 3) for MBP binding to DR(alpha, beta 1*0102)]. Although the location of the binding pocket in each DR molecule compared to the DR (alpha, beta 1 *0101) crystal is expected to be similar and suggests a common extended DR binding motif, the present results suggest that the relative importance of individual peptide anchor residues and of the corresponding DR binding pockets will differ for each DR/peptide complex.

Cloning and nucleotide sequence of the N-acetylmuramidase M1-encoding gene from Streptomyces globisporus.

The gene (acm) encoding N-acetylmuramidase M1 (ACM) was cloned of Streptomyces globisporus ATCC No. 21553. The nucleotide sequence of the acm gene was determined and found to code for an ORF of 294 amino acids (aa). Comparison of aa sequence deduced from the acm gene with the N-terminal sequence of the extracellular enzyme suggests that ACM is synthesized with a 77-aa leader peptide. A comparison of the ACM aa sequence with the aa sequences of other proteins in the NBRF data base reveals that ACM has strong similarity to the N-O-diacetylmuramidase secreted by the fungus Chalaropsis.