Complete amino acid sequence ofan HLA-DR antigen ... - Europe PMC

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Communicated by Peter Reichard, March 4, 1982. ABSTRACT The complete ...... Edelman, G. M. (1970)Biochemistry 9, 3197-3205. 48. Campbell, D. G.
Proc. NatL Acad. Sci. USA Vol. 79, pp. 3687-3691, June 1982 Biochemistry

Complete amino acid sequence of an HLA-DR antigen-like f3 chain as predicted from the nucleotide sequence: Similarities with immunoglobulins and HLA-A, -B, and -C antigens (cDNA clone/amino acid sequence homology/major histocompatibility complex/gene duplication)

DAN LARHAMMAR, LENA SCHENNING, KENTH GUSTAFSSON, KLAs WIMAN, LENA CLAESSON, LARS RASK, AND PER A. PETERSON Department of Cell Research, The Wallenberg Laboratory, University of Uppsala, Box 562, S-751 22 Uppsala, Sweden

Communicated by Peter Reichard, March 4, 1982

corresponding to the heavy chain of class I antigens (20-23), a prerequisite for obtaining primary structure information at a rapid rate. Consequently, we and others have isolated cDNA clones corresponding to the a (24, 25) and ,B chains (26) of HLADR-like antigens. In this communication, we provide the complete nucleotide sequence of one of the P-chain cDNA clones and demonstrate that the predicted amino acid sequence is homologous to -both class I antigen subunits and immunoglobulin chains.

ABSTRACT The complete nucleotide sequence of an HLADR antigen-like (3-chain cDNA clone was determined. The 1,080 base pairs include the complete coding region and most of the untranslated portion. The predicted amino acid sequence has 229 residues. The .8 chain contains two immunoglobulin-like disulfide loops and a 21-amino acid residue membrane-integrated segment. Ten amino acid residues reside on the cytoplasmic side of the plasma membrane. The single asparagine-linked carbohydrate moiety is attached to asparagine-19. The NH2-terminal 91 residues of the (3 chain are homologous to the corresponding region of HLA-A, -B, and -C antigen heavy chains. Residues 92-192 of the ( chain display statistically significant homology to members of the immunoglobulin family, P2-microglobulin, and the immunoglobulin-like domain of HLA-A, -B, and -C antigen heavy chains. These data establish that the major histocompatibility antigens of class I and class II type and the constant regions of immunoglobulins are evolutionarily related.

MATERIALS AND METHODS Enzymes and Chemicals. T4 polynucleotide kinase and restriction endonucleases were purchased from New England BioLabs. DNA polymerase I was a product of Boehringer Mannheim. [ y-32P]ATP (>400 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels) was obtained from Amersham. Plasmid pDR-f8-1. The construction and identification of pDR-/3-1 has been described (26). Plasmid DNA used to construct a restriction site map and to elucidate the nucleotide sequence was purified by CsCl/ethidium bromide equilibrium gradient centrifugation followed by sucrose gradient centrifugations (27, 28). Restriction Map of pDR-,8-1. The restriction map for pDR,B31 was constructed according to the procedure of Smith and Birnstiel (29) after labeling the BamHI site followed by cleavage with Sal I. Nucleotide Sequence Determinations. Highly purified pDR/-1 was digested with one of several endonucleases, end labeled with [y-32P]ATP (30), and cleaved with a second restriction endonuclease. Oligonucleotide fragments labeled in one 5' end were isolated by polyacrylamide gel electrophoresis. Separated DNA fragments were recovered from the gels by electroelution into bags of dialysis tubing. Nucleotide sequence determinations were carried out according to the procedures of Maxam and Gilbert (30) and Maat and Smith (31). Separation of the resulting oligonucleotide mixtures was carried out by electrophoresis under denaturing conditions in polyacrylamide gels of6%, 8%, and 20% acrylamide with multiple sample applications (30). Statistical Analyses for Relatedness of the Predicted pDR,8-1 Amino Acid Sequence to Other Proteins. The amino acid sequence predicted from the nucleotide sequence of the pDR/3-1 insert was compared with amino acid sequences of a large number of proteins. These sequences were maintained in a protein sequence data file (32) and comparisons were made using the SEARCH program (33). An input matrix, the mutation data matrix (250 PAM) (34), and a matrix bias parameter of 2 were supplied to the program. Homologies between the pDR/3-1 sequence and those of HLA-A, -B, and -C antigen chains and IgG constant and variable domains were analyzed by using

The major histocompatibility complex encompasses genes that control two types of cell surface-expressed transplantation antigens (see ref. 1). The class I antigens, termed HLA-A, -B, and -C antigens in man and H-2 K, D, and L in the mouse, are integral membrane proteins displaying extensive genetic polymorphism (2). They are composed of one invariant chain, /32microglobulin, and one heavy chain (3, 4). The heavy chain spans the lipid bilayer and has three extracellular domains, each having 90 amino acid residues (for review, see ref. 5). The second domain and the domain attached to the membrane-spanning segment contain immunoglobulin-like disulfide loops (6). However, only the latter domain is homologous in primary structure to immunoglobulin constant domains (7-11). /32-Microglobulin is also evolutionarily related to the immunoglobulin chains (12). The class II molecules, termed HLA-DR and Ia-antigens in man and mouse, respectively, are composed of dissimilar subunits, called a and /3 chains (13), both of which are integral membrane proteins that leave their COOH-terminal regions on the cytoplasmic side of the plasma membrane (14, 15). Class II antigens have been implicated in a variety ofimmunoregulatory events (16-18). Their role in the presentation of foreign antigens to T-helper cells has received much attention (see ref. 19). In the latter context, it appeared of interest to examine the primary structure of class II molecules to find out whether these molecules, like the class I antigens, display amino acid sequence homology with immunoglobulins. Recently, several groups have succeeded in cloning cDNA The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. ยง1734 solely to indicate this fact.

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