cDNA cloning of human plasminogen activator-inhibitor from endothelial cells.

Full-length cDNA for plasminogen activator inhibitor (PAI-1) was isolated from a human umbilical vein endothelial cell (HUVEC) lambda gt11 cDNA library. Three overlapping clones were identified by immunologic screening of 10(6) recombinant phage using a rabbit anti-human fibrosarcoma PAI-1 antiserum. The fusion proteins encoded by these three clones also react strongly with a monoclonal mouse anti-human fibrosarcoma PAI-1 antibody. By nucleotide sequence analysis, PAI-1 cDNA encodes a protein containing 402 amino acids with a predicted, nonglycosylated molecular mass of 45 kD. Identity of this material as authentic PAI-1 was confirmed by the presence of high level homology with the primary amino acid sequence of an internal peptide prepared from purified rat hepatoma PAI-1. The predicted amino acid sequence also reveals extensive homology with other members of the serine protease inhibitor gene family. Cultured HUVECs contain two PAI-1 mRNA species, both encoded by a single gene, differing by 1 kb in the 3' untranslated region. The PAI-1 gene is located on human chromosome 7.     

Isolation and expression of cDNA clones encoding a human receptor for IgG (Fc gamma RII)

We have cloned and expressed a cDNA encoding a human receptor for IgG (Fc gamma R) from the monocyte cell line U937. The deduced structure is a 35-kD transmembrane protein with homology to the mouse Fc[gamma 2b/gamma 1] receptor amino acid sequence of approximately 60% in the extracellular domain. The signal sequence is homologous to the mouse Fc gamma R alpha cDNA clone, while the transmembrane domain shares homology with mouse Fc gamma R beta cDNAs. The cytoplasmic domain is apparently unique. The extracellular domain shows significant homology to proteins of the Ig gene superfamily, including the human c-fms protooncogene/CSF-1 receptor. Mouse Ltk- cells transfected with the human Fc gamma R cDNA express a cell-surface receptor that selectively binds human IgG and is recognized by the anti-Fc gamma RII mAb IV.3. Antibodies against peptides derived from the human Fc gamma R sequence specifically stain U937 cells, but not an Fc gamma RII-bearing B-lymphoblastoid cell line (Daudi). These results identify the human Fc gamma RII as the homologue of mouse Fc[gamma 2b/gamma 1] R, and provide evidence for heterogeneity of Fc gamma RII expressed on monocytes and B cells.     

Localization of the gene for coagulation factor XIII a-chain to chromosome 6 and identification of sites of synthesis.

Factor XIII, the clotting factor essential for covalent stabilization of the fibrin clot, is a heterodimer consisting of a2 and b2 subunits, with catalytic function residing in the a-chain. In order to address questions regarding sites of synthesis and chromosomal localization of the Factor XIII a-chain, cDNA was cloned from a lambda gt11 human placental cDNA library. Nucleotide and amino acid sequences were determined from the cDNA. Amino acid sequencing of purified platelet Factor XIII a-chains confirmed the authenticity of the lambda gt11 clone. The gene for Factor XIII a-chain was mapped uniquely to chromosome 6. Northern blot analysis of human placental and U937 (monocytelike) cell poly (A)+ mRNA showed a single approximately 4.0-kb message for the Factor XIII a-chain. These results provide conclusive evidence that the a-chain is synthesized by placenta and monocyte cell lines.     

Molecular cloning, chromosomal localization, and functional characterization of a human liver Na+/bile acid cotransporter.

We have used a cDNA probe from a cloned rat liver Na+/taurocholate cotransporting polypeptide (Ntcp) to screen a human liver cDNA library. A 1,599-bp cDNA clone that encodes a human Na+/taurocholate cotransporting polypeptide (NTCP) was isolated. The human NTCP consists of 349 amino acids (calculated molecular mass of 38 kD) and exhibits 77% amino acid homology with the rat Ntcp. In vitro translation experiments indicate that the protein is glycosylated and has a molecular weight similar to the rat Ntcp. Injection of in vitro transcribed cRNA into Xenopus laevis oocytes resulted in the expression of Na(+)-dependent taurocholate uptake. Saturation kinetics indicated that the human NTCP has a higher affinity for taurocholate (apparent Km = 6 microM) than the previously cloned rat protein (apparent Km = 25 microM). NTCP-mediated taurocholate uptake into oocytes was inhibited by all major bile acid derivatives (100 microM), bumetanide (500 microM), and bromosulphophthalein (100 microM). Southern blot analysis of genomic DNA from a panel of human/hamster somatic cell hybrids mapped the human NTCP gene to chromosome 14.     

Maple syrup urine disease. Complete primary structure of the E1 beta subunit of human branched chain alpha-ketoacid dehydrogenase complex deduced from the nucleotide sequence and a gene analysis of patients with this disease.

A defect in the E1 beta subunit of the branched chain alpha-ketoacid dehydrogenase (BCKDH) complex is one cause of maple syrup urine disease (MSUD). In an attempt to elucidate the molecular basis of MSUD, we isolated and characterized a 1.35 kbp cDNA clone encoding the entire precursor of the E1 beta subunit of BCKDH complex from a human placental cDNA library. Nucleotide sequence analysis revealed that the isolated cDNA clone (lambda hBE1 beta-1) contained a 5'-untranslated sequence of four nucleotides, the translated sequence of 1,176 nucleotides and the 3'-untranslated sequence of 169 nucleotides. Comparison of the amino acid sequence predicted from the nucleotide sequence of the cDNA insert of the clone with the NH2-terminal amino acid sequence of the purified mature bovine BCKDH-E1 beta subunit showed that the cDNA insert encodes for a 342-amino acid subunit with a Mr = 37,585. The subunit is synthesized as the precursor with a leader sequence of 50 amino acids and is processed at the NH2 terminus. A search for protein homology revealed that the primary structure of human BCKDH-E1 beta was similar to the bovine BCKDH-E1 beta and to the E1 beta subunit of human pyruvate dehydrogenase complex, in all regions. The structures and functions of mammalian alpha-ketoacid dehydrogenase complexes are apparently highly conserved. Genomic DNA from lymphoblastoid cell lines derived from normal and five MSUD patients, in whom E1 beta was not detected by immunoblot analysis, gave the same restriction maps on Southern blot analysis. The gene has at least 80 kbp.     

The isolation and sequence of a novel gene from a human functional T cell line

Using a subtractive hybridization procedure we have constructed a cDNA library enriched for sequences present in functional human T cell lines, but not in human EBV-transformed B cell lines. We have isolated a cDNA clone, AH2-519, representing a novel gene, designated 519. This novel gene is expressed in functional human cytolytic and Th cell lines but not in a variety of other cell lines, including several long-term human T cell tumor lines. The expression of gene 519 is inducible in cultures of normal human PBL using antigenic or mitogenic stimulation. Neither the DNA sequence determined from a full-length cDNA clone overlapping with clone AH2-519 nor the amino acid sequence of its predicted protein product has significant homology to published sequences in the GenBank or NBRF databases. The restricted expression of gene 519 suggests that its gene product is involved in the growth and/or differentiation of normal T cells. The data also show that normal, nontransformed, functional T cells express gene products that can not be readily identified in long-term tumor lines of the same cell lineage.     

Structure of the human B lymphocyte receptor for C3d and the Epstein- Barr virus and relatedness to other members of the family of C3/C4 binding proteins [published erratum appears in J Exp Med 1988 Nov 1;168(5):1953-4]

Human complement receptor type 2 (CR2) is the B lymphocyte receptor for C3d and the Epstein-Barr virus. This protein is also a member of a family of C3b/C4b binding proteins that regulate complement activation, comprise tandemly repeated 60-75 amino acid sequences, and whose genes map to band q32 on chromosome 1. Overlapping cDNA clones encoding the entire human CR2 protein have been isolated from a human tonsillar cDNA library. The derived amino acid sequence of 1,032 residues encodes a peptide of 112,716 mol wt. A signal peptide was identified, followed by 15 copies of the short consensus repeat (SCR) structure common to the C3/C4 binding protein family. The entire extracellular portion of the protein comprised SCRs, thus, the ligand binding sites both for C3d and the EBV protein gp350/220 are positioned within this structure. Immediately following the final SCR was a transmembrane sequence of 24 amino acids and a cytoplasmic region of 34 amino acids. One of five cDNA clones isolated contained an additional SCR, providing evidence for alternative mRNA splicing or gene products of different human alleles. The CR2 cDNAs were used to isolate CR2-specific genomic phage. The entire CR2 coding sequences were found within 20 kb of human DNA. Analysis of the CR2 cDNA sequence indicated that CR2 contained internally homologous regions and suggested that CR2 arose by duplication of a primordial gene sequence encoding four SCRs. Comparison of the CR2 peptide sequence with those of other members of the gene family has identified many regions highly homologous with human CR1, fewer with C4bp and decay accelerating factor, and very few with factor H, and suggested that CR2 and CR1 arose by duplication of the same ancestral gene sequence. The homology between CR2 and CR1 extended to the transmembrane and cytoplasmic regions, suggesting that these sequences were derived from a common membrane-bound precursor.     

HLA DRB4 0101-restricted immunodominant T cell autoepitope of pyruvate dehydrogenase complex in primary biliary cirrhosis: evidence of molecular mimicry in human autoimmune diseases

We established six T cell clones specific for pyruvate dehydrogenase complex (PDC)-E2 peptides from four different patients with primary biliary cirrhosis using 33 different peptides of 17-20 amino acid residues corresponding to human PDC-E2 as stimulating antigens. The minimal T cell epitopes of these six T cell clones were all mapped to the same region of the PDC-E2 peptide 163-176 (GDLLAEIETDKATI), which corresponds to the inner lipoyl domain of PDC-E2. The HLA restriction molecules for this epitope were all identified as HLA DRB4 0101. The common essential amino acids of this epitope for these T cell clones were E, D, and K at positions 170, 172, and 173, respectively; other crucial amino acids for this epitope differed in each T cell clone. In addition, the alanine-substituted peptides at positions 170 and 173, but not 172, inhibited the proliferation of all T cell clones induced by the original peptide of human PDC-E2 163-176, indicating that amino acid D at position 172 is a critical MHC-binding site for all T cell clones tested. Interestingly, all T cell clones reacted to PDC-E2 peptide 36-49 (GDLIAEVETDKATV), which corresponds to the outer lipoyl domain of human PDC-E2. Furthermore, one T cell clone cross-reacted with exogenous antigens such as Escherichia coli PDC-E2 peptide 31- 44/134-147/235-248 (EQSLITVEGDKASM), which has an EXDK sequence. This is a definite demonstration of the presence of molecular mimicry at the T cell clonal level in human autoimmune diseases. It is also considered possible to design peptide-specific immunotherapy based on the findings of T cell autoepitopes in primary biliary cirrhosis.     

Serum vitamin D-binding protein is a third member of the albumin and alpha fetoprotein gene family.

A near full-length cDNA encoding the human vitamin D-binding protein (hDBP) was isolated from a human liver mRNA expression library. Complete sequence analysis of this clone predicts the full-length amino acid sequence of the pre-hDBP. Comparison of the sequence of the hDBP mRNA and protein to existing protein and nucleic acid data banks demonstrates a strong and highly characteristic homology of the hDBP with human albumin (hALB) and human alpha-fetoprotein (hAFP). Based upon this structural comparison, we establish that DBP is a member of the ALB and AFP gene family.     

Antigen mimicry in autoimmune disease sharing of amino acid residues critical for pathogenic T cell activation.

A nonamer peptide from murine nicotinic acetylcholine receptor delta chain (ACR delta), which shared four amino acid residues with a nonamer peptide of murine ovarian zona pellucida glycoprotein ZP3, induced murine autoimmune oophoritis and IgG autoantibody to the zona pellucida. Crossreaction between the ACR delta and ZP3 peptides was established by the response of a ZP3 peptide-specific, oophoritogenic T cell clone to both peptides in association with IA (alpha k beta b). By substituting the ZP3 peptides with a single alanine, four amino acids within the ZP3 peptide were found to be important for ovarian autoimmune disease, autoantibody response, and stimulation of the ZP3-specific T cell clone. Substitution with conservative amino acids of three residues also ablated activity, whereas the fourth, a phenylalanine, was replaceable by tyrosine without loss of activity. Of the four critical amino acids, three were shared between the ZP3 peptide and the ACR delta peptide. Moreover, polyalanine peptides with the four critical ZP3 amino acids or the four amino acids common to the ZP3 and ACR delta peptides induced immune response to ZP3 and elicited severe ovarian autoimmune disease. Thus, organ-specific autoimmune disease can occur through immune response against unrelated self (or foreign) peptides that share with a self-peptide sufficient common amino acid residues critical for activation of pathogenic, autoreactive T cells.     

Cloning and chromosomal localization of a human kidney cDNA involved in cystine, dibasic, and neutral amino acid transport.

We have recently cloned, sequenced, and characterized a rat kidney cDNA (D2) that stimulates cystine as well as dibasic and neutral amino acid transport. In order to evaluate the role of this protein in human inherited diseases such as cystinuria, we have isolated a human D2 clone (D2H) by low stringency screening of a human kidney cDNA library using the radiolabeled D2 insert as a probe. The D2H cDNA is 2284 nucleotides long and encodes a 663 amino acid protein that is 80% identical to the rat D2 amino acid sequence and 86% to that of the rabbit homologue rBAT. Microinjection of in vitro transcribed D2H cRNA into Xenopus oocytes induced uptake of cystine as well as dibasic and neutral amino acids in a pattern similar to that of rat D2 and rabbit rBAT. Both neutral and dibasic amino acids inhibited the D2H-induced uptake of cystine. Northern blot analysis demonstrated that D2H, like D2 and rBAT, is expressed strongly in the kidney and intestine. Southern blot analysis of genomic DNA from a panel of mouse-human somatic cell hybrids showed that the human gene for D2H resides on chromosome 2.     

Cloning and characterization of a pyridoxine 5'-phosphate oxidase from silkworm, Bombyx mori

A cDNA encoding Pyridoxine 5'-phosphate oxidase (PNPO) from Bombyx mori was cloned and characterized (G en B ank accession number: DQ452398). The cDNA encodes a polypeptide of 257 amino acid residues. The recombinant enzyme purified from Escherichia coli exhibited maximal activity at pH 9.0, and the K _m values for the substrates of pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate were determined as 0.65 and 1.15 µmol/l. It was found that B. mori PNPO shares 51.44% homology with humans, but several function-related, key amino acid residues in B. mori PNPO are different from the human and E. Coli gene. B. mori has a single copy of the PNPO gene, which spans a 3.5 kb region and contains five exons and four introns. B. mori PNPO is a homodimer, with each monomer containing nine antiparallel β-strands and five α-helical segments. The secondary structure was deduced from computational study.     

Genes encoding pancreatic polypeptide and neuropeptide Y are on human chromosomes 17 and 7.

Pancreatic polypeptide and neuropeptide Y share 50% amino acid homology (18 out of 36 residues), suggesting that they may have common ancestral origins. cDNA clones complementary to human mRNAs encoding pancreatic polypeptide and neuropeptide Y were used to detect specific human genomic DNA sequences in human-mouse somatic cell hybrid lines. The pancreatic polypeptide gene (PPY) segregated with human chromosome 17, while the neuropeptide Y gene (NPY) segregated with human chromosome 7. Examination of cell hybrids with chromosomal rearrangements assigned PPY to the p11.1-qter region and NPY to the pter-q22 region of their respective chromosomes.     

Molecular cloning and sequence of the complementary DNA encoding human mitochondrial acetoacetyl-coenzyme A thiolase and study of the variant enzymes in cultured fibroblasts from patients with 3-ketothiolase deficiency.

Complementary DNAs encoding the precursor of human hepatic mitochondrial acetoacetyl-CoA thiolase (T2) (EC 2.3.1.9) were cloned and sequenced. The cDNA inserts in these clones were 1,518 bases in length when overlapped, and encoded the 427-amino acid precursor of this enzyme (45,199 mol wt). This amino acid sequence included a 33-residue leader peptide moiety and a 394-amino acid subunit of the mature enzyme (41,385 mol wt). The T2 gene expression in fibroblasts from four patients with 3-ketothiolase deficiency was analyzed by Northern blotting. The T2 mRNA in all four cell lines had the same 1.7 kb as that of the control. However, the amounts of T2 mRNA differed: the content was reduced in two cell lines (cases 1 and 3), whereas it was within a normal range in others (cases 2 and 4). Pulse labeling followed by subcellular fractionation revealed that the T2 proteins in the fibroblasts from these patients are present in the mitochondria. These results suggest that different mechanisms are involved in the enzyme defects in the four patients.     

Molecular cloning, expression, and chromosomal localization of the human earliest lymphocyte activation antigen AIM/CD69, a new member of the C-type animal lectin superfamily of signal-transmitting receptors

The activation of T lymphocytes, both in vivo and in vitro, induces the expression of CD69. This molecule, which appears to be the earliest inducible cell surface glycoprotein acquired during lymphoid activation, is involved in lymphocyte proliferation and functions as a signal transmitting receptor in lymphocytes, natural killer (NK) cells, and platelets. To determine the structural basis for CD69 function, the cDNA coding for CD69 was isolated by a polymerase chain reaction-based strategy using oligonucleotides deduced from peptide sequences of the purified protein. The isolated cDNA exhibited a single open reading frame of 597 bp coding for CD69, and predicted a 199-amino acid protein of type II membrane topology, with extracellular (COOH-terminal), transmembrane, and intracellular domains. The CD69 clone hybridized to a 1.7-kb mRNA species, which was rapidly induced and degraded after lymphocyte stimulation, consistent with the presence of rapid degradation signals at the 3' untranslated region. Transient expression of the polypeptide encoded by CD69 cDNA in COS-7 cells demonstrated that it presented properties comparable to native CD69 protein. The CD69 gene was regionally mapped to chromosome 12 p13-p12 by both somatic cell hybrid DNA analysis and fluorescence in situ hybridization coupled with GTG banding (G bands by trypsin using Giemsa). Protein sequence homology search revealed that CD69 is a new member of the Ca(2+)-dependent (C-type) lectin superfamily of type II transmembrane receptors, which includes the human NKG2, the rat NKR-P1, and the mouse NKR-P1 families of NK cell-specific genes. CD69 also has a structural homology with other type II lectin cell surface receptors, such as the T cell antigen Ly49, the low avidity immunoglobulin E receptor (CD23), and the hepatic asialoglycoprotein receptors. The CD69 protein also shares functional characteristics with most members of this superfamily, which act as transmembrane signaling receptors in early phases of cellular activation.     

De novo generation of cationic antimicrobial peptides: influence of length and tryptophan substitution on antimicrobial activity

Comparison of human immunodeficiency virus lentiviral lytic peptide 1 with other host-derived peptides indicates that antimicrobial properties of membrane-active peptides are markedly influenced by their cationic, hydrophobic, and amphipathic properties. Many common themes, such as Arg composition of the cationic face of an amphipathic helix and the importance of maintaining the hydrophobic face, have been deduced from these observations. These studies suggest that a peptide with these structural properties can be derived de novo by using only a few strategically positioned amino acids. However, the effects of length and helicity on antimicrobial activity and selectivity have not been objectively evaluated in the context of this motif. To address these structure-function issues, multimers of a 12-residue lytic base unit (LBU) peptide composed only of Arg and Val residues aligned to form idealized amphipathic helices were designed. Bacterial killing assays and circular dichroism analyses reveal a strong correlation between antibacterial activity, peptide length, and propensity to form a helix in solvent mimicking the environment of a membrane. Increasing peptide length beyond two LBUs (24-residue peptides) resulted in no appreciable increase in antimicrobial activity. Derivatives (WLBU) of the LBU series were further engineered by substituting Trp residues in the hydrophobic domains. The 24-residue WLBU2 peptide was active at physiologic NaCl concentrations against Staphylococcus aureus and mucoid and nonmucoid strains of Pseudomonas aeruginosa. Further, WLBU2 displayed the highest antibacterial selectivity of all peptides evaluated in the present study by using a coculture model of P. aeruginosa and primary human skin fibroblasts. These findings provide fundamental information toward the de novo design of an antimicrobial peptide useful for the management of infectious diseases.