Molecular cloning and expression of mouse GD1alpha/GT1aalpha/GQ1balpha synthase (ST6GalNAc VI) gene

A novel member of the mouse CMP-NeuAc:beta-N-acetylgalactosaminide alpha2,6-sialyltransferase (ST6GalNAc) subfamily, designated ST6GalNAc VI, was identified by BLAST analysis of expressed sequence tags. The sequence of the cDNA clone of ST6GalNAc VI encoded a type II membrane protein with 43 amino acids composing the cytoplasmic domain, 21 amino acids composing the transmembrane region, and 269 amino acids composing the catalytic domain. The predicted amino acid sequence showed homology to the previously cloned ST6GalNAc III, IV, and V, with common amino acid sequences in sialyl motif L and S among these four enzymes. A fusion protein with protein A and extracts from L cells transfected with ST6GalNAc VI in an expression vector showed enzyme activity of alpha2,6-sialyltransferase for GM1b, GT1b, and GD1a but not toward glycoproteins. Thin layer chromatography-immunostaining revealed that the products were GD1alpha, GQ1balpha, and GT1aalpha. Northern blotting revealed that this gene was expressed in a wide range of mouse tissues such as colon, liver, heart, spleen, and brain. It is concluded that this enzyme is a novel sialyltransferase involved in the synthesis of alpha-series gangliosides in the nervous tissues and many other tissues.     

Isolation and characterization of CA XIV, a novel membrane-bound carbonic anhydrase from mouse kidney.

Carbonic anhydrase (CA) is involved in various physiological processes such as acid-base balance and transport of carbon dioxide and ions. In this study, we have succeeded in the isolation of a novel CA from the mouse kidney by use of the signal sequence trap method. It is a 337-amino acid polypeptide with a calculated molecular mass of 37.5 kDa, consisting of a putative amino-terminal signal sequence, a CA domain, a transmembrane domain, and a short hydrophilic carboxyl terminus, which we designated CA XIV. The CA domain of CA XIV is highly homologous with those of known CAs, especially extracellular CAs including CA XII, IX, VI, and IV. The expression study of an epitope-tagged protein has suggested that CA XIV is located on the plasma membrane. When expressed in COS-7 cells, CA XIV exhibits CA activity that is predominantly associated with the membrane fraction. By Northern blot analysis, the gene expression of CA XIV is most abundant in the kidney and heart, followed by the skeletal muscle, brain, lung, and liver. In situ hybridization has revealed that, in the kidney, the gene is expressed intensely in the proximal convoluted tubule, which is the major segment for bicarbonate reabsorption and also in the outer border of the inner stripe of the outer medulla. In conclusion, we have cloned a functional cDNA encoding a novel membrane-bound CA. This study will bring new insights into our understanding of carbon dioxide metabolism and acid-base balance.     

Cloning of a cDNA encoding an Artemia franciscana Na/K ATPase alpha-subunit.

Clones of cDNA that code for an isoform of the Artemia franciscana Na/K ATPase alpha subunit (NaKA alpha) have been isolated. The sequence of the longest of these clones (pArATNa136) is 3595 nucleotides; it codes for a 1004-amino acid protein whose sequence is identical to that of two previously sequenced Artemia NaKA alpha peptides. The encoded protein is over 73% identical to Drosophila melanogaster and vertebrate NaKA alpha s, and 73.8% identical to another Artemia NaKA alpha isoform previously described (named alpha 2850 in this article). The two Artemia cDNA clones code for mRNAs of different size; the clone pArATNa136 codes for a 4.5-kb mRNA while the alpha 2850 clone codes for a 3.6-kb mRNA. The degree of homology and the different size of the mRNAs encoded by both cDNAs suggest that they code for two different isoforms of the protein.     

Identification of a novel secretory leukocyte protease inhibitor-binding protein involved in membrane phospholipid movement

Previous studies have suggested that human salivary secretory leukocyte protease inhibitor (SLPI) inhibits HIV-1 by binding to a host cell surface protein of unknown identity. Using the yeast two-hybrid assay, we identified a gene sequence encoding a novel SLPI-binding protein (SLPI-BP). The 1.5-kb cDNA encodes a 318-amino acid protein with a predicted transmembrane segment near the C-terminus. Sequence analysis revealed that SLPI-BP is the human scramblase protein that is involved in the movement of membrane phospholipids. Co-expression of SLPI and SLPI-BP followed by an S-protein pulldown assay confirmed the specific interaction between these two proteins. Our data represent the first report for the identity of SLPI-BP.     

Identification of a novel annexin in Hydra vulgaris. Characterization, cDNA cloning, and protein kinase C phosphorylation of annexin XII.

As a first step toward the elucidation of a simple animal model in which to investigate annexin function, we identified, isolated, and characterized a novel annexin from Hydra vulgaris, annexin XII. A hydra cDNA library was screened using a probe generated by polymerase chain reaction from primers based on the partial amino acid sequence of annexin XII. Annexin XII cDNA was cloned and the functional protein was expressed in high yields in Escherichia coli. The annexin XII cDNA sequence predicted a 316-amino acid protein that had between 44 and 54% sequence identity with the Ca2+-binding core domains of previously characterized vertebrate and Drosophila annexins. The amino-terminal domain of annexin XII did not have sequence similarity with other known annexins except at and around a site that resembled known protein kinase C (PKC) phosphorylation sites in other annexins. As anticipated from its sequence, annexin XII was a high affinity substrate for purified rat brain PKC; half-maximal phosphorylation occurred below 0.1 microM annexin XII, and incorporation of up to 0.8 mol of phosphate/mol of annexin XII was observed. A PKC-like activity in hydra extracts also phosphorylated annexin XII. In summary, hydra promises to be a valuable model system for investigating the biological function of annexins and for determining how this function is modulated by PKC phosphorylation.     

The primary structure of the VLA-2/collagen receptor alpha 2 subunit (platelet GPIa): homology to other integrins and the presence of a possible collagen-binding domain

VLA-2 (also called gpIa/IIa on platelets) is a collagen receptor with a unique alpha subunit and a beta subunit common to other adhesion receptors in the VLA/integrin family. Multiple cDNA clones for the human VLA-2 alpha 2 subunit have been selected from a lambda gtll library by specific antibody screening. The 5,374-bp nucleotide sequence encoded for 1,181 amino acids, including a signal peptide of 29 amino acids followed by a long extracellular domain (1,103 amino acids), a transmembrane domain, and a short cytoplasmic segment (22 amino acids). Direct sequencing of purified alpha 2 protein confirmed the identity of the 15 NH2-terminal amino acids. Overall, the alpha 2 amino acid sequence was 18-25% similar to the sequences known for other integrin alpha subunits. In particular, the alpha 2 sequence matched other integrin alpha chains in (a) the positions of 17 of its 20 cysteine residues; (b) the presence of three metal-binding domains of the general structure DXDXDGXXD; and (c) the transmembrane domain sequence. In addition, the alpha 2 sequence has a 191-amino acid insert (called the I-domain), previously found only in leukocyte integrins of the beta 2 integrin family. The alpha 2 I-domain was 23-41% similar to domains in cartilage matrix protein and von Willebrand factor, which are perhaps associated with collagen binding. The NH2-terminal sequence reported here for alpha 2 does not match the previously reported alpha 2 NH2-terminal sequence (Takada, Y., J. L. Strominger, and M. E. Hemler. 1987. Proc. Natl. Acad. Sci. USA. 84:3239-3243). Resolution of this discrepancy suggests that there may be another VLA heterodimer that resembles VLA-2 in size but has a different amino acid sequence.     

Complete amino acid sequence of a novel integrin beta subunit (beta 6) identified in epithelial cells using the polymerase chain reaction

The integrin family of adhesion receptors consists of several heterodimeric glycoproteins, each composed of one alpha and one beta subunit. Three different mammalian beta subunits, beta 1, beta 2, and beta 3, have been sequenced, but recent evidence suggests the existence of several others. Amplification of guinea pig airway epithelial cell cDNA with oligonucleotide primers designed to recognize consensus integrin beta subunit sequences led to the identification of a novel partial cDNA sequence. Clones containing portions of this sequence were used to screen cDNA libraries constructed from the human pancreatic carcinoma cell line FG-2 and identified a series of overlapping clones encoding the full-length sequence of the human homologue of this protein. This sequence of 788 amino acids is 43, 38, and 47% identical to the sequences of beta 1, beta 2, and beta 3, respectively. Features shared between this novel protein and the previously sequenced beta subunits include the positions of all 56 cysteine residues in the extracellular domain, the single putative transmembrane domain, and the short putative cytoplasmic domain. However, a unique 11-amino acid extension at the carboxyl terminus, not present in any of the other beta subunits, is suggestive of distinctive interactions with cytoplasmic components. Comparison of the human and guinea pig sequences reveals a high degree (94%) of cross-species conservation. Because this protein is clearly distinct from the two other recently described integrins beta 4 and beta 5, we propose to designate it beta 6.     

An ABC transporter homologous to TAP proteins.

Polymerase chain reaction amplification of cDNA from rat intestine revealed the expression of a novel ABC transporter, TAPL (TAP-like). Subsequently, the protein sequence was deduced from the nucleotide sequence of cDNA carrying the entire coding region. TAPL is transcribed ubiquitously in various rat tissues. The protein, with 762 amino acid residues, has potential transmembrane domains, and an ATP-binding domain in its amino and carboxyl terminal regions, respectively, and is highly homologous to TAP1 and TAP2 (transporters associated with antigen presentation/processing): pairwise comparisons with TAPL demonstrated 39 and 41% of the residues are identical, respectively. These numerical values are essentially the same as that for TAP1 and TAP2 (39%), and the hydropathy profiles of TAPL, TAP1 and TAP2 are quite similar. The similarity among these three proteins suggests that they could be derived from a common ancestral gene. Furthermore, we found that there is a potential splicing isoform, sharing the amino terminal 720 amino acid residues of TAPL.     

Molecular cloning of a novel alpha2,3-sialyltransferase (ST3Gal VI) that sialylates type II lactosamine structures on glycoproteins and glycolipids

A novel member of the human CMP-NeuAc:beta-galactoside alpha2, 3-sialyltransferase (ST) subfamily, designated ST3Gal VI, was identified based on BLAST analysis of expressed sequence tags, and a cDNA clone was isolated from a human melanoma line library. The sequence of ST3Gal VI encoded a type II membrane protein with 2 amino acids of cytoplasmic domain, 32 amino acids of transmembrane region, and a large catalytic domain with 297 amino acids; and showed homology to previously cloned ST3Gal III, ST3Gal IV, and ST3Gal V at 34, 38, and 33%, respectively. Extracts from L cells transfected with ST3Gal VI cDNA in a expression vector and a fusion protein with protein A showed an enzyme activity of alpha2, 3-sialyltransferase toward Galbeta1,4GlcNAc structure on glycoproteins and glycolipids. In contrast to ST3Gal III and ST3Gal IV, this enzyme exhibited restricted substrate specificity, i.e. it utilized Galbeta1,4GlcNAc on glycoproteins, and neolactotetraosylceramide and neolactohexaosylceramide, but not lactotetraosylceramide, lactosylceramide, or asialo-GM1. Consequently, these data indicated that this enzyme is involved in the synthesis of sialyl-paragloboside, a precursor of sialyl-Lewis X determinant.     

A new member of the protein kinase C family, nPKC theta, predominantly expressed in skeletal muscle.

A new protein kinase C (PKC)-related cDNA with unique tissue distribution has been isolated and characterized. This cDNA encodes a protein, nPKC theta, which consists of 707 amino acid residues and showed the highest sequence similarity to nPKC delta (67.0% in total). nPKC theta has a zinc-finger-like cysteine-rich sequence (C1 region) and a protein kinase domain sequence (C3 region), both of which are common in all PKC family members. However, nPKC theta lacks a putative Ca2+ binding region (C2 region) that is seen only in the conventional PKC subfamily (cPKC alpha, -beta I, -beta II, and -gamma) but not in the novel PKC subfamily (nPKC delta, -epsilon, -zeta, and -eta). Northern (RNA) blot analyses revealed that the mRNA for nPKC theta is expressed predominantly in skeletal muscle. Furthermore, nPKC theta mRNA is the most abundantly expressed PKC isoform in skeletal muscle among the nine PKC family members. nPKC theta expressed in COS1 cells serves as a phorbol ester receptor. By the use of an antipeptide antibody specific to the D2-D3 region of the nPKC theta sequence, nPKC theta was recognized as a 79-kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis in mouse skeletal muscle extract and also in an extract from COS1 cells transfected with an nPKC theta cDNA expression plasmid. Autophosphorylation of immunoprecipitated nPKC theta was observed; it was enhanced by phosphatidylserine and 12-O-tetradecanoylphorbol-13-acetate but attenuated by the addition of Ca2+. These results clearly demonstrate that nPKC theta should be considered a member of the PKC family of proteins that play crucial roles in the signal transduction pathway.     

Molecular cloning of mouse protein kinase C (PKC) cDNA from Swiss 3T3 fibroblasts

A set of complementary DNAs (cDNAs) that encode the complete 672-amino acid sequence for mouse protein kinase C (PKC) type alpha have been isolated from a mouse Swiss 3T3 cDNA library. Extensive rescreening of this cDNA library only resulted in the isolation of clones of the same PKC species, indicating that Swiss 3T3 fibroblasts express exclusively PKC-alpha. This enzyme is encoded by two different mRNAs that exhibit a substantial heterogeneity in their 3'-noncoding regions. This heterogeneity could have been derived from alternative splicing of the pre-mRNAs or be due to differential usage of the polyadenylation motif. The expression of PKC mRNA in fibroblasts is very low and it is not influenced by treatment with the tumor promoter 12-O-tetradecanoyl-13-phorbol-acetate.     

Amino acid sequence of the alpha subunit of human leukocyte adhesion receptor Mo1 (complement receptor type 3)

Mo1 (complement receptor type 3, CR3; CD11b/CD18) is an adhesion-promoting human leukocyte surface membrane heterodimer (alpha subunit 155 kD [CD11b] noncovalently linked to a beta subunit of 95 kD [CD18]). The complete amino acid sequence deduced from cDNA of the human alpha subunit is reported. The protein consists of 1,136 amino acids with a long amino-terminal extracytoplasmic domain, a 26-amino acid hydrophobic transmembrane segment, and a 19-carboxyl-terminal cytoplasmic domain. The extracytoplasmic region has three putative Ca2+-binding domains with good homology and one with weak homology to the "lock washer" Ca2+-binding consensus sequence. These metal-binding domains explain the divalent cation-dependent functions mediated by Mo1. The alpha subunit is highly homologous to the alpha subunit of leukocyte p150,95 and to a lesser extent, to the alpha subunit of other "integrin" receptors such as fibronectin, vitronectin, and platelet IIb/IIIa receptors in humans and position-specific antigen-2 (PS2) in Drosophila. Mo1 alpha, like p150, contains a unique 187-amino acid stretch NH2-terminal to the metal-binding domains. This region could be involved in some of the specific functions mediated by these leukocyte glycoproteins.     

Isolation and characterization of nudC from mouse macrophages, a gene implicated in the inflammatory response through the regulation of PAF-AH(I) activity

We report the characterization of a cDNA induced in mouse macrophages that encodes a 332-amino acid protein with extensive sequence identity with members of the mammalian nudC-like genes. The interaction between mNUDC and the regulatory beta subunit of platelet activating factor acetylhydrolase I (PAF-AH(I)) shown in this article indicates a new function of NUDC. Thus, we show that NUDC increases the catalytic activity of PAF-AH(I) and that this regulatory activity is located in the carboxyl terminal half of the protein which is highly conserved. This suggests a novel function for mammalian nudC-like genes as anti-inflammatory proteins.     

Cloning, Golgi localization, and enzyme activity of the full-length heparin/heparan sulfate-glucuronic acid C5-epimerase

While studying the cellular localization and activity of enzymes involved in heparan sulfate biosynthesis, we discovered that the published sequence for the glucuronic acid C5-epimerase responsible for the interconversion of d-glucuronic acid and l-iduronic acid residues encodes a truncated protein. Genome analysis and 5'-rapid amplification of cDNA ends was used to clone the full-length cDNA from a mouse mastocytoma cell line. The extended cDNA encodes for an additional 174 amino acids at the amino terminus of the protein. The murine sequence is 95% identical to the human epimerase identified from genomic sequences and fits with the general size and structure of the gene from Drosophila melanogaster and Caenorhabditis elegans. Full-length epimerase is predicted to have a type II transmembrane topology with a 17-amino acid transmembrane domain and an 11-amino acid cytoplasmic tail. An assay with increased sensitivity was devised that detects enzyme activity in extracts prepared from cultured cells and in recombinant proteins. Unlike other enzymes involved in glycosaminoglycan biosynthesis, the addition of a c-myc tag or green fluorescent protein to the highly conserved COOH-terminal portion of the protein inhibits its activity. The amino-terminally truncated epimerase does not localize to any cellular compartment, whereas the full-length enzyme is in the Golgi, where heparan sulfate synthesis is thought to occur.     

Retinoic acid-specific induction of a protein kinase C isoform during differentiation of HL-60 cells.

Human promyelocytic leukemia cells (HL-60) were treated with several differentiation inducers, then the changes in the activity of cytosolic protein kinase C (PKC) isoforms were examined by hydroxylapatite chromatography and the species of the isoforms were determined immunologically. In three undifferentiated HL-60 cell lines examined, PKC alpha and beta isoforms were present, but PKC gamma isoform was not detected. When the cells were induced by dimethylsulfoxide, dibutyryl cAMP, or nicotinamide to differentiate into granulocytes, these two PKC isoforms each increased to about 2- to 3-fold. When retinoic acid was used as the inducer, in addition to PKC alpha and beta, a third PKC isoform appeared. This isoform was clearly distinct from rat PKC alpha, beta, and gamma, immunologically. This isoform showed a distinctly lower Ca(2+)-requirement (3 microM) than that of PKC alpha or beta (100 microM) and was more dependent on cardiolipin and phosphatidylethanolamine, compared with PKC alpha, beta, and gamma. These results suggest that while the increases in the activities of PKC alpha and beta isoforms are common in the differentiation program initiated by several inducers, including retinoic acid, the emergence of an unclassified PKC isoform is a retinoic acid-specific process.     

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.