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Biomedical subjects

J F Kaufman

Publications and source records attributed to J F Kaufman.

At least 19 recordsLinked to original sources
PubMed1989

The major histocompatibility complex in the chicken.

The chicken B complex is the first non-mammalian MHC characterized at the molecular level. It differs from the human HLA and murine H-2 complexes in the small size of the class I (B-F) and class II (B-L) genes and their close proximity. This proximity accounts for the absence of recombination between B-F and B-L genes and leaves no space for class III genes. Moreover the B-F and B-L genes are tightly linked to unrelated genes absent from mammalian MHCs, such as the polymorphic B-G genes and a member of the G protein beta subunit family. This linkage could form the basis for resistance to viral-induced tumors associated with some B complex haplotypes.

Animals↗
PubMed1988

Two cell surface proteins bind the sponge Microciona prolifera aggregation factor.

Two extracellular matrix cell surface proteins which bind the proteoglycan-like aggregation factor from the marine sponge Microciona prolifera (MAF) and which may function as physiological receptors for MAF were identified and characterized for the first time. By probing nitrocellulose blots of nonreducing sodium dodecyl sulfate gels containing whole sponge cell protein with iodinated MAF, a 210- and a 68-kDa protein, which have native molecular masses of approximately 200-400 and 70 kDa, were identified. MAF binding to blots is species-specific. It is also sensitive to reduction and is completely abolished by pretreatment of live cells with proteases, as was cellular aggregation, indicating that the 210- and 68-kDa proteins may be located on the cell surface. The additional observations that the 68 kDa is an endoglycosidase F-sensitive glycoprotein and that antisera against whole sponge cells or membranes can immunoprecipitate the 210 kDa when prebound to intact cells are consistent with a cell surface location. Both proteins can be isolated from sponge cell membranes and from the sponge skeleton (insoluble extracellular matrix), but the 210-kDa MAF-binding protein can also be found in the soluble extracellular matrix (buffer washes of cells and skeleton) as well. A third MAF-binding protein of molecular mass 95 kDa was also found in the sponge extracellular matrix but rarely on cells. Both of the cell-associated 210- and 68-kDa proteins are nonintegral membrane proteins, based on Triton X-114 phase separation, flotation of liposomes containing sponge membrane lysates, and their extraction from membranes by buffer washes. Both proteins bind MAF affinity resins, indicating that they each exhibit a moderate affinity for MAF under native conditions. They can also be separated from each other and from the bulk of the protein in an octylpolyoxyethylene extract of membranes by fast protein liquid chromatography Mono Q anion exchange chromatography, as assessed by native dot blot and denaturing Western blot assays. Although neither protein bound to heparin, gelatin, hexosamine, or uronic acid-Sepharose resins, their affinity for an invertebrate proteoglycan, their roles in sponge cell adhesion, and their peripheral membrane protein natures suggest that they may represent early invertebrate analogs of cell-associated vertebrate extracellular matrix adhesion proteins, such as fibronectin or vitronectin, or else an entirely novel set of cell adhesion molecules.

Animals↗
PubMed1986

Major histocompatibility complex-encoded class I molecules are absent in immunologically competent Xenopus before metamorphosis.

The expression of class I and class II major histocompatibility complex (MHC)-encoded antigens has been examined at various stages of the development of the clawed frog, Xenopus. By immunoprecipitation with alloantisera or xenoantisera from radio-labeled spleen and thymus lysates, and by mixed lymphocyte reaction analysis, it was determined that the same class II molecules are expressed throughout ontogeny. In contrast, by fluorescence on frozen sections of tadpoles and by immunoprecipitation, the class I molecule is not detected in tadpoles, but appears on all tissues at the climax of metamorphosis. Animals maintained as tadpoles for long periods of time by chemical treatment do express class I antigens; thus, their expression can be independent of other biochemical and morphological changes that occur at metamorphosis. Immunofluorescence detects an otherwise uncharacterized MHC-linked alloantigen on tadpole thymic epithelium from the earliest stages of thymus differentiation.

Animals↗
PubMed1985

Xenopus MHC class II molecules. I. Identification and structural characterization.

Class II antigens from the Xenopus laevis MHC (f haplotype) were identified by using a rabbit antihuman class II beta-chain serum (anti-p29boost). This xenoantiserum inhibits bidirectional Xenopus MLR (but not PHA-stimulation), recognizes the same molecules as certain MHC-linked Xenopus alloantisera, and immunoprecipitates class II molecules from Xenopus cells consistent with the tissue distribution of mammalian class II molecules. The Xenopus class II molecules are composed of two different chains, both of which are 30 to 35kD transmembrane glycoproteins. The alpha-chains have some N-terminal sequence homology with mammalian class II alpha-chains (both I-E/DR and I-A/DC); the beta-chains are directly recognized by anti-p29boost and have a markedly increased SDS gel mobility under nonreducing conditions. During biosynthesis, they are noncovalently associated with a number of other chains, including ones at 25kD, 33kD, and 40 to 45kD. The alpha-chains bear three N-linked glycans (two Endo H insensitive in mature material) and the beta-chains bear two (one Endo H insensitive). Unlike most mammalian class II molecules, the deglycosylated beta-chains are significantly larger and more acidic than the alpha-chains.

Animals↗
PubMed1985

Xenopus MHC class II molecules. II. Polymorphism as determined by two-dimensional gel electrophoresis.

The class II antigens from four inbred strains of Xenopus laevis (r, f, g, and j haplotypes) and six gynogenetic LG clones (two Xenopus laevis, two Xenopus gilli haplotypes) with functionally well-defined MHC types have been immunoprecipitated with the rabbit anti-human class II beta-chain serum anti-p29boost and analyzed by two-dimensional gel electrophoresis. The glycosylated material from 15-hr biosynthetically labeled cells runs as a broad fuzzy band around 33kD that, upon removal of N-linked glycans by Endo F, resolves into upper beta-chain bands and lower alpha-chain bands. Both the glycosylated and deglycosylated class II antigens give rise to multiple IEF spots in evenly spaced arrays (alpha-chain: two to eight spots in one to three arrays, beta-chain: two to 12 spots in one to five arrays). Both chains are polymorphic and both map to the functionally defined MHC. The large number of spots argues for multiple class II antigens; by radioactive N-terminal sequencing, two homologous alpha-chains and five beta-chains are present in the f haplotype. By comparison with MHC-linked alloantisera, anti-p29boost recognizes all major polymorphic class II molecules in Xenopus laevis. A selection of outbred animals were typed by using an IEF procedure requiring only a million PHA-stimulated blood cells.

Animals↗
PubMed1984

Cysteines in the transmembrane region of major histocompatibility complex antigens are fatty acylated via thioester bonds.

Exogenous radioactive palmitic acid is incorporated post-translationally into the HLA-B and -DR heavy chains, but not HLA-A heavy chains or -DR light chains of the human B lymphoblastoid cells JY and T51 . Protease digestions localize the label to the transmembrane region of the B7 heavy chain. Both B7 and DR heavy chains have a cysteine in the transmembrane hydrophobic region, while the A2 heavy chain and DR light chains have none. Palmitic acid is covalently linked to these transmembrane cysteines via a thioester bond since: 1) the label is not removed by organic extraction or boiling in sodium dodecyl sulfate and dithiothreitol, but is released at room temperature by methanolic KOH as methyl palmitate, and by hydroxylamine as palmitohydroxamate . 2) The pH sensitivity and kinetics of release by hydroxylamine and Tris are similar to those of palmitoyl-CoA (thioester linkage) and unlike those of methyl palmitate and palmitoyllysophosphatidylcholine ( hydroxyester linkages). 3) Neutral hydroxylamine treatment (but not neutral Tris treatment) generates sites that can be reduced and alkylated in the transmembrane region of B7 heavy chain and to a lesser extent in DR heavy chain. 4) Organic extraction of pronase digests of labeled B7 yields peptides containing palmitate and cysteine (but not serine or threonine) which co-migrate by thin layer chromatography. A population of beta 2-microglobulin molecules not associated with heavy chains is palmitylated , but not via a thioester linkage.

Cysteine↗
PubMed1984

Identification of class I major histocompatibility complex encoded molecules in the amphibian Xenopus.

Class I-like molecules have been immunoprecipitated from Xenopus leukocytes and erythrocytes with alloantisera directed against major histocompatibility complex (MHC)-linked antigens. The heavy chains, depending on the allele examined, have molecular weights of 40 000-44 000 of which 3000 daltons are asparagine-linked carbohydrates, probably present as one N-linked glycan. The presumed analogue of beta 2-microglobulin has a molecular weight of 13 000 and bears no asparagine-linked glycans. Family studies show that the heavy chains are encoded by genes residing in or closely linked to the MHC.

Alleles↗
PubMed1983

The extracellular region of light chains from human and murine MHC class II antigens consists of two domains.

The experiments in this report, together with previous studies, demonstrate that the light chains of DR antigens are composed of four domains: two large extracellular domains (each with a disulfide loop), a short hydrophobic membrane-binding region, and a short hydrophilic carboxy-terminal domain. The amino-terminal extracellular domain is glycosylated and polymorphic and has no discernible sequence homology to immunoglobulin, whereas the relatively conserved carboxy-terminal extracellular domain bears striking homology to immunoglobulin. One chymotryptic and two tryptic cleavage sites lie in the second extracellular domain of the light chain of all native DR antigens. These three sites are found in loops at the same end of an immunoglobulin-like domain. The light chain of DC1 antigen lacks one of the tryptic cleavage sites; the DR heavy chain, the class I heavy chain, and beta 2 microglobulin lack all three proteolytic sites. Proteolysis of murine la antigens with chymotrypsin and trypsin generates similar fragments (I-E-like DR, I-A-like DC1) suggesting these cleavage sites are a general feature of class II antigen light chains.

Amino Acid Sequence↗
PubMed1982

cDNA clones for the heavy chain of HLA-DR antigens obtained after immunopurification of polysomes by monoclonal antibody.

A monoclonal antibody (HC 2.1) directed against the separated heavy chain of HLA-DR has been prepared. By binding HC 2.1 to polysomes from human B lymphoblastoid cells followed by the use of a protein A-Sepharose column as an immunoadsorbent, we have purified the mRNA coding for the HLA-DR heavy chain nearly to homogeneity. The immunopurified mRNA has been used to prepare labeled cDNA with which to probe cDNA libraries. Double-stranded cDNA was also made from the immunopurified mRNA and cloned directly into pBR322. Two clones, one from each of the above procedures, positively selected DR heavy chain message as assayed by cell-free translation and immunoprecipitation. One clone, pDRH-2 [500 base pairs plus 75 base pairs of poly(A)] contains the entire 3' untranslated region as well as coding information for the carboxy-terminal hydrophilic intracellular domain and part of the hydrophobic transmembrane region. Results of carboxypeptidase digestion of the heavy chains from detergent-solubilized (p34) and papain-treated (p33) HLA-DR antigen were consistent with the predicted protein sequence. Specific immunopurification of polysomes by defined monoclonal antibodies followed by direct cloning of cDNA to the highly purified mRNA is a powerful method for obtaining identified cDNA clones.

Amino Acid Sequence↗
PubMed1980

Specificity of mouse cytotoxic T lymphocytes stimulated with either HLA-A and -B or HLA-DR antigens reconstituted into phospholipid vesicles.

Distinct populations of murine cytolytic T lymphocytes (CTL) were elicited from primed spleen cells by preparations of HLA-A and -B (HLA-A,B) or HLA-DR antigens reconstituted into phospholipid vesicles. These populations could be distinguished by both antiserum blocking and by patterns of cytolysis of a panel of target cells. Cytolysis by CTL stimulated with liposomes that contained HLA-A2 and HLA-B7 antigens could only be blocked by antiserum against HLA-A,B antigens but not by antiserum against HLA-DR antigens. The inverse pattern was seen with HLA-DR-stimulated CTL. When compared with a panel of target cells expressing various HLA-A,B or HLA-DR allospecificities, the strongest CTL reactivity was seen toward those cells that bore the same allospecificities as those presented on the liposomes. Target cells that expressed cross-reactive specificities and unrelated specificities were recognized much less well. The implications of the results for the mechanism of CTL stimulation by liposomes, as well as the relationship between allogeneic and xenogeneic CTL recognition, are discussed.

Animals↗
PubMed1980

HLA-DR antigens have polymorphic light chains and invariant heavy chains as assessed by lysine-containing tryptic peptide analysis.

HLA-DR antigens were isolated from B lymphoblastoid cells labeled with either [3H]- or [14C]lysine. Double-label comparisons of the tryptic peptides by microbore ion-exchange chromatography led to the following conclusions: The heavy and light chains share few peptides, one of which is free lysine that is liberated from the polypeptide chains by trypsin. The heavy chains of different HLA-DR allospecificities are virtually identical in all cell lines examined. In contrast, the light chains of different HLA-DR allospecificities are nonidentical and, thus, must bear the alloantigenic determinant.

B-Lymphocytes↗
PubMed1980

Cell-free synthesis and processing of the heavy and light chains of HLA-DR antigens.

Antisera have been prepared against the separated glycoprotein chains (p29 and p34) of HLA-DR antigens (p29,34) isolated from membranes of the B lymphoblastoid cell line JY (DRw4,6). These antisera (anti-p29 and anti-p34) were characterized by immunoprecipitation of in vivo labeled, detergent-solubilized extracts of JY cells grown in the presence and absence of tunicamycin, an inhibitor of N-linked glycosylation. Anti-p29 and anti-p34 specifically immunoprecipitated the precursors to p29 and p34, respectively, from the cell-free translation products of a rabbit reticulocyte lysate system supplemented with polyadenylic acid-containing messenger RNA (mRNA) isolated from JY cells. These precursors, pre-p34 and two pre-p29s, appeared to be 1,500-3,000 daltons larger than their counterparts from tunicamycin-treated cells and presumably were synthesized with N-terminal extensions (signal sequences). Processing of pre-p29 and pre-p34 occurred during cell-free translation in the presence of dog pancreatic microsomes, which resulted in cleavage of the polypeptide, addition of glycan moieties, and segregation of the two chains in the cisternal portion of the microsome. the precursors of p29 and p34 were not appreciably different in isoelectric points on two-dimensional gels from p26 and p28, the light and heavy chains from antigens arise from separate mRNA, and not as a single polypeptide, which is endoproteolytically cleaved.

B-Lymphocytes↗
PubMed1979

Both chains of HLA-DR bind to the membrane with a penultimate hydrophobic region and the heavy chain is phosphorylated at its hydrophilic carboxyl terminus.

The HLA-DR antigen, a complex of two glycoproteins of 29,000 and 34,000 daltons, can be isolated from the membranes of human B-lymphoblastoid cell lines. Extensive proteolysis releases only 5-10% of the antigen, whereas detergent solubilizes all of it. Detergent solubilization after papain proteolysis of membranes produces antigen with chains cleaved near the carboxyl termini. Comparison of these three preparations demonstrates that each chain contains a carboxyl-terminal hydrophilic region that is sensitive to proteolytic degradation and a penultimate hydrophobic region, responsible for membrane binding, that is more resistant to papain. This two-step cleavage of each chain is also observed during the proteolysis of detergent-solubilized HLA-DR antigen. Both chains of HLA-DR in the membrane can be labeled with the lipophilic photoactivatable carbene reagent adamantane diazirine. This label is released from both chains during the second cleavage. The heavy chain can be reduced and alkylated under mild conditions, and this label is also lost during the second cleavage. The heavy chain is phosphorylated in vivo, and this label is lost upon the first cleavage. This observation suggests that the carboxyl terminus of the heavy chain is intracellular. Cumulatively, these data suggest that both chains of HLA-DR antigens are comprised of large extracellular NH2-terminal regions, small penultimate intramembranous regions, and small carboxyl-terminal intracellular regions.

Amino Acid Sequence↗