Cloning and expression of the cDNA coding for a human lymphocyte IgE receptor.

Low-affinity receptors (Fc epsilon R) and secreted factors (IgE-BF) which bind to immunoglobulins of the IgE isotype play a key role in the regulation of human IgE synthesis. We report here the cloning of a cDNA coding for the Fc epsilon R of the human B-lymphoblast cell line RPMI 8866. The nucleotide sequence of this cDNA predicts a polypeptide with 321 amino acids and a mol. wt of 36,281 daltons. A functional Fc epsilon R capable of binding IgE was expressed in Chinese hamster ovary cells after stable transformation with the cDNA which had been cloned into a mammalian expression vector. Amino acid sequence analysis of IgE-BF purified from RPMI 8866 cells revealed an amino-terminal sequence of 19 residues which coincides with the predicted amino acid sequence of the Fc epsilon R, starting at residues 148 and 150. A computer search with the translated amino acid sequence of the Fc epsilon R revealed a domain of 120 amino acids having striking homology to the human asialoglycoprotein receptors.     

Molecular cloning and sequence analysis of cDNA encoding the macrophage lectin specific for galactose and N-acetylgalactosamine

The primary structure of the macrophage lectin specific for galactose and N-acetylgalactosamine (macrophage asialoglycoprotein-binding protein, M-ASGP-BP) has been deduced from its cDNA sequence. The M-ASGP-BP cDNA encoded a protein consisting of 306 amino acid residues with a molecular mass of 34,242 daltons. The sequence was highly homologous with that of the rat liver asialoglycoprotein receptor (rat hepatic lectin, RHL), particularly that of RHL-1 (the major form of RHL), throughout its whole length, and especially so in its putative membrane-spanning region and carbohydrate recognition domain. There were two N-glycosylation sites in M-ASGP-BP, the location of which were identical to those in RHL-1. However, M-ASGP-BP was characteristic in having a shorter cytoplasmic tail, and an inserted segment of 24 amino acids containing an Arg-Gly-Asp sequence between the membrane-spanning region and carbohydrate recognition domain.     

Mouse asialoglycoprotein receptor cDNA sequence: conservation of receptor genes during mammalian evolution

The asialoglycoprotein receptor internalizes galactose-terminated glycoproteins into mammalian hepatocytes for degradation in lysosomes. We report the cloning and sequencing of one murine asialoglycoprotein receptor cDNA which exhibits homology with rat and human receptor forms. Conserved regions may correlate with functional domains. The membrane-bound M (mouse) HL polypeptide does not contain a cleavable N-terminal signal sequence and is probably anchored to the membrane via an internal insertion sequence.     

Cloning and sequence analysis of a cDNA encoding human non-selective type of endothelin receptor.

A cDNA encoding non-selective type (ETB) of endothelin receptor was isolated from a human liver cDNA library. The cDNA had an open reading frame encoding a protein of 442 amino acid residues with a relative Mr of 49,643. The deduced amino acid sequence of human ETB receptor was 88% and 64% identical to those of rat lung ETB receptor and bovine lung ET-1-specific (ETA) receptor, respectively, and contained a relatively long and proline-rich extracellular N-terminal region in addition to a significant similarity with the G protein-coupled receptor super-family with seven transmembrane segments.     

Molecular cloning and expression of the cDNA for alpha 3 subunit of human alpha 3 beta 1 (VLA-3), an integrin receptor for fibronectin, laminin, and collagen

alpha 3 beta 1 (VLA-3), a member of the integrin family of cell adhesion receptors, may function as a receptor for fibronectin, laminin, and collagen. A partial cDNA clone (2.4 kb) for the human alpha 3 subunit was selected from an endothelial cell lambda gt11 cDNA library by specific antibody screening. Several overlapping cDNA clones were subsequently obtained, of a total length of 4.6 kb from various cDNA libraries. The reconstructed alpha 3 cDNA was expressed on the surface of chinese hamster ovary cells as detected by an alpha 3- specific mAb after transfection, suggesting that the cDNA is authentic. Within this sequence was an open reading frame, encoding for 1,051 amino acids, including a signal peptide of 32 residues, a long extracellular domain (959 residues), a transmembrane domain (28 residues), and a short cytoplasmic segment (32 residues). Overall, the alpha 3 amino acid sequence was 25-37% similar to the other integrin alpha subunits that are cleaved, with most similarity to the alpha 6 sequence (37%), and less similarity to those alpha subunits that have I domains (15-20%, excluding the I domain sequence itself). Features most like those in other alpha subunits are (a) the positions of 18/19 cysteine residues, (b) three potential metal binding domains of the general structure DX(D/N)X(D/N)GXXD, and (c) the predicted transmembrane domain. The mass of alpha 3 calculated from its amino acid sequence is 113,505. The human alpha 3 sequence was 89% identical to hamster galactoprotein b3, and 70% similar to the chicken CSAT antigen band 2 protein partial sequence, suggesting that these two polypeptides are homologues of human alpha 3.     

Primary structure of the mannose receptor contains multiple motifs resembling carbohydrate-recognition domains

Macrophages express a cell surface receptor which mediates phagocytosis and pinocytosis of particles and solutes containing mannose (fucose and N-acetylglucosamine are also ligands for the receptor). An apparently identical protein has been isolated from human placenta. Proteolytic fragments of the placental receptor were sequenced so that oligonucleotide probes complementary to the receptor cDNA could be generated. These probes were used to isolate cDNA clones covering the entire coding portion of the mRNA for the receptor. Confirmation that these clones encode the mannose receptor was obtained by expression in rat fibroblasts. The expressed protein mediates uptake and degradation of mannose-conjugated serum albumin. The deduced amino acid sequence of the receptor reveals that it is most likely to be a type I transmembrane protein (COOH terminus on the cytoplasmic side of the membrane) since the mature polypeptide is preceded by a signal sequence and a hydrophobic stop transfer sequence is located 45 amino acids from the COOH terminus. The extracellular portion of the receptor polypeptide consists of three types of domains. The first 139 amino acids constitute a cysteine-rich segment which does not resemble other known sequences. There follows a domain which closely resembles fibronectin type II repeats. The remainder of the extracellular portion of the receptor is composed of eight segments homologous with the C-type carbohydrate-recognition domains of the asialoglycoprotein receptor, mannose binding proteins, and other Ca2(+)-dependent animal lectins. This structure suggests that the receptor may contain multiple ligand-binding domains thus accounting for its tight binding to highly multivalent ligands.     

Gene transfer into hepatocytes using asialoglycoprotein receptor mediated endocytosis of DNA complexed with an artificial tetra-antennary galactose ligand.

We have constructed an artificial ligand for the hepatocyte-specific asialoglycoprotein receptor for the purpose of generating a synthetic delivery system for DNA. This ligand has a tetra-antennary structure, containing four terminal galactose residues on a branched carrier peptide. The carbohydrate residues of this glycopeptide were introduced by reductive coupling of lactose to the alpha- and epsilon-amino groups of the two N-terminal lysines on the carrier peptide. The C-terminus of the peptide, containing a cysteine separated from the branched N-terminus by a 10 amino acid spacer sequence, was used for conjugation to 3-(2-pyridyldithio)propionate-modified polylysine via disulfide bond formation. Complexes containing plasmid DNA bound to these galactose-polylysine conjugates have been used for asialoglycoprotein receptor-mediated transfer of a luciferase gene into human (HepG2) and murine (BNL CL.2) hepatocyte cell lines. Gene transfer was strongly promoted when amphipathic peptides with pH-controlled membrane-disruption activity, derived from the N-terminal sequence of influenza virus hemagglutinin HA-2, were also present in these DNA complexes. Thus, we have essentially borrowed the small functional domains of two large proteins, asialoglycoprotein and hemagglutinin, and assembled them into a supramolecular complex to generate an efficient gene-transfer system.     

Cloning and characterization of the cDNAs for human and rabbit interleukin-1 precursor.

DNA sequence complementary to the mRNA for rabbit interleukin-1 precursor (preIL-1) has been cloned from the cDNA library constructed using partially purified poly(A)+RNA from induced rabbit alveolar macrophages by mRNA hybridization-translation assay. By using this cDNA as a probe, human IL-1 cDNA was isolated from the cDNA library prepared using poly(A)+RNA from induced HL-60 cells, a human monocyte-like cell line. The amino acid sequences of the human and rabbit preIL-1 deduced from the cDNA sequences reveal their primary structures which consists of 271 and 267 amino acid residues, respectively. The amino acid sequence is 64% conserved between human and rabbit. The difference in number of amino acid residues results from the carboxy-terminal extention of 4 amino acid residues in human preIL-1. Expression of the cloned human cDNA in E. coli yielded biologically active IL-1.     

Cloning of a Drosophila cDNA encoding a polypeptide similar to the human insulin receptor precursor

A Drosophila cDNA clone was obtained using the human insulin receptor cDNA sequence as a probe. The 3586 bp nucleotide sequence predicted a single polypeptide of 1095 amino acid residues which showed considerable homology (35.2%) with the human insulin receptor precursor. Although the cDNA was incomplete at its 5'-terminal region, it encodes a transmembrane glycoprotein as a single precursor of a two subunit molecule having a structural architecture similar to that of the human insulin receptor precursor. The presumptive beta subunit carries a well conserved Tyr kinase domain which showed 63.5% homology with that of human insulin receptor; however the protein of the alpha subunit is only weakly conserved (25%).     

Molecular cloning, sequence analysis and expression of a cDNA encoding human type-1 angiotensin II receptor.

We isolated a cDNA encoding type-1 angiotensin II receptor from a human liver cDNA library. The cDNA had an open reading frame encoding a protein of 359 amino acid residues with a relative Mr of 41,060. The deduced amino acid sequence of the human angiotensin II (Ang II) receptor was 95.3% and 94.2% identical to those of bovine and rat type-1 Ang II receptors, respectively, and had a significant similarity with the G protein-coupled receptor. The rank order of the binding to the receptor expressed in COS-7 cells was Ang II greater than Ang III greater than Ang I. The expression of the Ang II receptor mRNA was detected in human liver, lung, adrenal and adrenocortical adenomas but not in adrenomedullary tumor, pheochromocytoma, by Northern blot analysis.     

Plasma cell membrane glycoprotein PC-1. Primary structure deduced from cDNA clones

The PC-1 protein is a membrane glycoprotein that is selectively expressed on the surface of antibody-secreting cells. Previous work has shown that it consists of two apparently identical disulfide-bonded polypeptides, each of molecular weight approximately 120,000. We now describe the sequence of PC-1 mRNA and protein. The PC-1 protein is shown to consist of 905 amino acids and to have an uncommon transmembrane orientation. The NH2-terminal 58 residues are intracellular and the COOH-terminal 826 residues are extracellular. A cysteine-rich region of 85 amino acids lies adjacent to the extracellular surface of the membrane and appears to have arisen by exon duplication. In common with other membrane glycoproteins with this orientation, there is no obvious signal sequence other than the transmembrane segment. The PC-1 protein therefore has an overall structure and membrane orientation that resembles those of the transferrin receptor, the asialoglycoprotein receptor, and the Ia invariant chain.     

Molecular cloning and sequencing of a cDNA for a carbohydrate binding receptor unique to rat Kupffer cells

cDNA comprising the entire length of the rat Kupffer cell receptor (Mr = 88,000 and 77,000) for carbohydrates with an affinity for fucose and galactose was isolated, and its nucleotide sequence was determined. Receptor cDNA encoded a protein containing 550 amino acid residues with a Mr = 61,104. This Mr was consistent with the size of the deglycosylated receptor which was found to contain two polypeptides by gel electrophoresis with Mr = 58,000 and 52,000, respectively. Edman degradation of the receptor yielded a sequence which corresponded to amino acid residues 83-104 of the sequence derived from the cDNA. This confirmed that the cDNA which had been isolated corresponded to mRNA for the receptor and suggested that the smaller polypeptide in receptor preparations arises by proteolysis of the intact receptor. Amino acid composition of the receptor was nearly identical to that predicted by the cDNA. The Kupffer cell receptor was found to be homologous to other carbohydrate binding proteins including the hepatic receptors with different binding specificities. The Kupffer cell receptor also contained a series of 18 contiguous, homologous sequences with an average length of 14 residues.     

Cloning and sequencing of a rat type II activin receptor.

A full-length cDNA for a rat type II activin receptor was cloned by hybridization from a rat ovary cDNA library. The deduced amino acid sequence (513 residues) containing a single membrane-spanning domain and an intracellular kinase domain with predicted serine/threonine specificity. The amino acid sequence is 99.8% and 99.4% identical in the coding region with the previously cloned mouse and human type II activin receptor, and only 66.7% identical in the coding region with the previously cloned rat type IIB activin receptor. We examined the effect of PMSG-hCG on the mRNA level of type II activin receptor in immature rat ovaries. Northern blot analysis of ovarian RNA revealed two mRNAs (3.0 kb and 6.0 kb).     

Cloning and sequence analysis of cDNAs encoding mammalian mitochondrial malate dehydrogenase

A cDNA clone, named ppmMDH-1 and covering a part of the porcine mitochondrial malate dehydrogenase (mMDH; L-malate:NAD+ oxidoreductase, EC 1.1.1.37) mRNA, was isolated from a porcine liver cDNA library with a mixture of 24 oligodeoxyribonucleotides as a probe. The sequences of the probe were deduced from the known sequence of porcine mMDH amino acid residues 288-293. ppmMDH-1 covered the coding region for porcine mMDH amino acid residues 17-314 and the 3' untranslated region. Subsequently, mouse mMDH cDNA clones were isolated from a mouse liver cDNA library with the ppmMDH-1 cDNA as a probe. One of the clones, named pmmMDH-1 and containing a cDNA insert of about 1350 base pairs, was selected for sequence analysis, and the primary structure of the mouse precursor form of mMDH (pre-mMDH) was deduced from its cDNA sequence. The sequenced coding regions for the porcine and mouse mMDH mRNAs showed about 85% homology. When the deduced amino acid sequence of the mouse pre-mMDH was compared with that of the porcine mMDH, they shared a 95% homology, and the mouse pre-mMDH yielded a leader sequence consisting of 24 amino acid residues and a mature mMDH, consisting of 314 amino acid residues. The leader sequence contained three basic amino acid residues, no acidic residues, and no hydrophobic amino acid stretch. The mouse mMDH leader sequence was compared with those of three other rodent mitochondrial matrix proteins.     

Amino- and carboxy-terminal sequence of mouse J chain and analysis of tryptic peptides

Mouse J chain was isolated from an IgM-producing hybridoma by gel filtration and ion-exchange chromatography. The sequence of the amino-terminal 25 residues was determined. At these positions, the results agree with the amino acid sequence deduced from the cDNA sequence determined previously by Koshland and co-workers and indicate that a leader sequence terminating in glycine is removed to form the mature J chain. Tryptic peptides of J chain were isolated by high pressure liquid chromatography and their amino acid compositions were compared with those expected from the cDNA sequence. The amino acid sequence of the carboxy-terminal peptide and a mixture of two other peptides was determined. The results were consistent with the cDNA sequence except that we found valine, not leucine, at position 67, and arginine, not glycine, at position 117. The presence of aspartic acid at the carboxy-terminus, as predicted from the cDNA, indicates that processing does not occur at this end of the polypeptide chain. Upon amino acid analysis, glucosamine was found in tryptic peptides 47-57 and 47-58. J chain was also cleaved at aspartylproline bonds with formic acid and the unfractionated digest was subjected to automated Edman degradation. The mixed sequence was consistent with the sequence deduced from the cDNA at positions 1 to 13, 28 to 40, 52 to 64, and 73 to 85. In conjunction with the results obtained previously by analysis of cDNA, these data show that mouse J chain is a polypeptide containing 137 amino acid residues, 93 of which are identical to residues in human J chain.     

Cloning and characterization of a new functional human aldehyde dehydrogenase gene

We cloned a new functional ALDH gene (ALDHx) from a human genomic library in cosmid pWE-15 by screening with a 29-nucleotide probe partially matched to a conserved region of the ALDH1 and ALDH2 genes. The new ALDHx gene does not contain introns in the coding sequence for 517 amino acid residues. The degree of resemblance between the deduced amino acid sequences of the new ALDHx gene and the ALDH2 gene is 72.5% (alignment of 517 amino acid residues), while that between the ALDHx and the ALDH1 gene is 64.6% (alignment of 500 amino acid residues). The amino acid residues (Cys-162, Cys-302, Glu-268, Glu-487, Gly-223, Gly-225, Gly-229, Gly-245 and Gly-250), which exist in both ALDH1 and ALDH2 isozymes and have been implicated in functional and structural importance, are also preserved in the deduced sequence of the new ALDHx gene. Northern blot hybridization with ALDHx probe revealed the existence of a unique mRNA band (3.0 kilobases) in the human liver and testis tissues. Using the new ALDHx probe, we cloned the cDNA of the gene from a human testis cDNA library in lambda gt11 vector. The nucleotide sequence of the cDNA differs from that of the genomic sequence at three nucleotide positions resulting in the exchange of 2 deduced amino acid residues. These positions are polymorphic as further demonstrated by the PCR amplification of the targeted region followed by nucleotide sequence analysis of the genomic DNA from eight unrelated individuals. Alignment of the genomic and cDNA sequence indicates that although the ALDHx gene appears to have no intron in its coding sequence, an intron of 2.6 kilobases is found to interrupt the 5'-untranslated (5'-UT) sequence. Primary extension and S1 mapping analysis indicate the existence of at least two 5'-UT exons. The new ALDHx gene was assigned to chromosome 9 by Southern blot hybridization of DNA samples from a panel of rodent-human hybrid cell lines.     

Deletion analysis of the internal signal-anchor domain of the human asialoglycoprotein receptor H1.

The human asialoglycoprotein receptor H1 is a single-spanning membrane protein with the amino terminus facing the cytoplasm and the carboxy terminus exposed on the exoplasmic side of the plasma membrane. It has been shown earlier that the transmembrane segment, residues 38-65, functions as an internal signal directing protein synthesis to the endoplasmic reticulum and initiating membrane insertion. This process is co-translational and mediated by signal recognition particle (SRP). To identify subsegments within this region containing the signal information, we prepared deletion mutants at the level of the cDNA and analysed them in a wheat germ in vitro translation system with microsomes as the target membrane. Insertion and membrane anchoring were judged by the glycosylation of the protein, its resistance to exogenous protease and the extent to which it can be extracted from the microsomes by alkaline treatment. It was found that very small deletions already reduce the stability of membrane anchoring. However, nearly half of the transmembrane domain can be deleted, both from the amino-terminal and from the carboxy-terminal side, without completely abolishing membrane insertion. Several mutants, although not inserted, still interact with SRP. The results support the notion that the main feature of a signal sequence is a hydrophobic stretch of sufficient length (10-12 residues in our sequence), and indicate that recognition by SRP is not sufficient for membrane insertion.     

The human LDL receptor: a cysteine-rich protein with multiple Alu sequences in its mRNA

The nucleotide sequence of a cloned 5.3 kilobase cDNA for the human low density lipoprotein receptor revealed five domains in the 839 amino acid protein: 322 NH2-terminal amino acids, extremely rich in disulfide-bonded cysteine residues (15%) and including an 8-fold repeat of 40 residues that may contain the LDL binding site; 350 residues homologous to the precursor of mouse epidermal growth factor; a region immediately outside the plasma membrane, rich in serine and threonine and the site of O-linked glycosylation; 22 hydrophobic amino acids, spanning the plasma membrane; and 50 COOH-terminal amino acids, projecting into the cytoplasm. The mRNA for the receptor contains a 3' untranslated region of 2.5 kilobases that includes multiple copies of the Alu family of repetitive DNAs. Transfection of simian COS cells with the human LDL receptor cDNA linked to the SV40 early promoter resulted in expression of functional cell surface receptors.