Cloning, characterization, and phylogenetic analysis of siglec-9, a new member of the CD33-related group of siglecs. Evidence for co-evolution with sialic acid synthesis pathways

The Siglecs are a subfamily of I-type lectins (immunoglobulin superfamily proteins that bind sugars) that specifically recognize sialic acids. We report the cloning and characterization of human Siglec-9. The cDNA encodes a type 1 transmembrane protein with three extracellular immunoglobulin-like domains and a cytosolic tail containing two tyrosines, one within a typical immunoreceptor tyrosine-based inhibitory motif (ITIM). The N-terminal V-set Ig domain has most amino acid residues typical of Siglecs. Siglec-9 is expressed on granulocytes and monocytes. Expression of the full-length cDNA in COS cells induces sialic-acid dependent erythrocyte binding. A recombinant soluble form of the extracellular domain binds to alpha2-3 and alpha2-6-linked sialic acids. Typical of Siglecs, the carboxyl group and side chain of sialic acid are essential for recognition, and mutation of a critical arginine residue in domain 1 abrogates binding. The underlying glycan structure also affects binding, with Galbeta1-4Glc[NAc] being preferred. Siglec-9 shows closest homology to Siglec-7 and both belong to a Siglec-3/CD33-related subset of Siglecs (with Siglecs-5, -6, and -8). The Siglec-9 gene is on chromosome 19q13.3-13.4, in a cluster with all Siglec-3/CD33-related Siglec genes, suggesting their origin by gene duplications. A homology search of the Drosophila melanogaster and Caenorhabditis elegans genomes suggests that Siglec expression may be limited to animals of deuterostome lineage, coincident with the appearance of the genes of the sialic acid biosynthetic pathway.     

Isolation and characterization of the gene for myosin light chain two of Drosophila melanogaster

A recombinant lambda-phage DNA clone containing Drosophila melanogaster sequences encoding the gene for myosin light chain (MLC) two has been isolated from a library of randomly sheared DNA. The Drosophila MLC2 gene is located in region 99E1-3 on the right arm of chromosome 3, several bands removed from the site reported for the other myosin light chain gene at 98B. The MLC2 sequence at 99E1-3 appears to encode all of the isoforms of Drosophila MLC2. The polypeptide encoded at 99E was identified as MLC2 by the following criteria: the in vitro translation product is identical in size to MLC2 isolated from Drosophila muscle, and on two-dimensional gels the in vitro translation product can be separated into two or more peptides that co-migrate with isoforms of larval and thoracic MLC2. RNA encoding the polypeptide was detected in embryos only after the onset of muscle differentiation and was also abundant in adult thoracic muscle. The nucleotide sequence of cDNA generated from late embryonic RNA would be translated to yield a protein sequence with multiple regions of homology to vertebrate MLC2. (There are shorter regions of homology to vertebrate MLC1). Like a number of vertebrate muscle proteins, Drosophila MLC2 has an acetylated amino-terminus.     

Cloning, expression and characterization of the pig liver GDP-mannose pyrophosphorylase. Evidence that GDP-mannose and GDP-Glc pyrophosphorylases are different proteins.

GDP-Man, the mannosyl donor for most Man-containing polymers is formed by the transfer of Man-1-P to GTP to form GDP-Man and PPi. This reaction is catalyzed by the widespread and essential enzyme, GDP-Man pyrophosphorylase (GMPP). The pig liver GMPP consists of an alpha subunit (43 kDa) and a beta subunit (37 kDa). Purified pig GMPP catalyzes the synthesis of GDP-Glc (from Glc-1-P and GTP) and GDP-Man (from Man-1-P and GTP), but has higher activity for the formation of GDP-Glc than for synthesis of GDP-Man. In the present study, we report the cloning of the cDNA for the beta subunit of GMPP, and its expression in a bacterial system resulting in the formation of active enzyme. The full length cDNA encoding the beta subunit was isolated from a porcine cDNA library, and its predicted gene product showed high amino-acid sequence homology to GMPPs from other species. The gene was expressed in Escherichia coli cells, and a 37-kDa protein was over-produced in these cells. This gene product reacted strongly with antibody reactive to the native beta subunit of pig GMPP. Most interestingly, this recombinant protein had high activity for synthesizing GDP-Man (from Man-1-P and GTP), but very low activity for the formation of GDP-Glc (from Glc-1-P and GTP). Other properties of the recombinant protein were also analyzed. This study suggests that the beta subunit is the GMPP, whereas the alpha subunit, or a combination of both subunits, may have the GDP-Glc pyrophosphorylase activity.     

Characterization of mammalian orthologues of the Drosophila osa gene: cDNA cloning, expression, chromosomal localization, and direct physical interaction with Brahma chromatin-remodeling complex

The osa gene of Drosophila melanogaster encodes a nuclear protein that is a component of the Brahma chromatin-remodeling complex. Osa is required for embryonic segmentation, development of the notum and wing margin, and photoreceptor differentiation. In these tissues, osa mutations have effects opposite to those caused by wingless (wg) mutations, suggesting that osa functions as an antagonist of wg signaling. Here we describe the cloning and characterization of mammalian orthologues of osa. Three evolutionarily conserved domains were identified in Osa family members: the N-terminal Bright domain and C-terminally located Osa homology domains 1 and 2. RNase protection analysis indicates a widespread expression of the Osa1 gene during mouse development, in adult tissues, and in cultured cell lines. The Osa1 gene was localized to mouse chromosome 4, within the region syntenic to chromosomal position 1p35-p36 of its human counterpart. We present evidence that the OSA1 product is localized in the nucleus and associates with human Brahma complex, which suggests evolutionarily conserved function for Osa in gene regulation between mammals and Drosophila.     

Isolation and characterization of a human cDNA for mRNA 5'-capping enzyme.

The human mRNA 5'-capping enzyme cDNA was identified. Three highly related cDNAs, HCE1 (human mRNAcappingenzyme1), HCE1A and HCE1B , were isolated from a HeLa cDNA library. The HCE1 cDNA has the longest ORF, which can encode a 69 kDa protein. A short region of 69 bp in the 3'-half of the HCE1 ORF was missing in HCE1A and HCE1B , and, additionally, HCE1B has an early translation termination signal, which suggests that the latter two cDNAs represent alternatively spliced product. When expressed in Escherichia coli as a fusion protein with glutathione S -transferase, Hce1p displayed both mRNA 5'-triphosphatase (TPase) and mRNA 5'-guanylyltransferase (GTase) activities, and it formed a cap structure at the 5'-triphosphate end of RNA, demonstrating that it indeed specifies an active mRNA 5'-capping enzyme. The recombinant proteins derived from HCE1A and HCE1B possessed only TPase activity. When expressed from ADH1 promoter, HCE1 but not HCE1A and HCE1B complemented Saccharomyces cerevisiae CEG1 and CET1 , the genes for GTase and TPase, respectively. These results demonstrate that the N-terminal part of Hce1p is responsible for TPase activity and the C-terminal part is essential for GTase activity. In addition, the human TPase domain cannot functionally substitute for the yeast enzyme in vivo.     

Analysis of a cDNA from the neurologically active locus shaking-B (Passover) of Drosophila melanogaster.

We have isolated and sequenced a cDNA from the shaking-B locus of Drosophila melanogaster. The cDNA contains an open reading frame with extensive homology to another D. melanogaster gene, l(1)ogre. This suggests the existence of a new family of proteins required for the development and maintenance of the D. melanogaster nervous system.     

PARP-2, A novel mammalian DNA damage-dependent poly(ADP-ribose) polymerase.

Poly(ADP-ribosylation) is a post-translational modification of nuclear proteins in response to DNA damage that activates the base excision repair machinery. Poly(ADP-ribose) polymerase which we will now call PARP-1, has been the only known enzyme of this type for over 30 years. Here, we describe a cDNA encoding a 62-kDa protein that shares considerable homology with the catalytic domain of PARP-1 and also contains a basic DNA-binding domain. We propose to call this enzyme poly(ADP-ribose) polymerase 2 (PARP-2). The PARP-2 gene maps to chromosome 14C1 and 14q11.2 in mouse and human, respectively. Purified recombinant mouse PARP-2 is a damaged DNA-binding protein in vitro and catalyzes the formation of poly(ADP-ribose) polymers in a DNA-dependent manner. PARP-2 displays automodification properties similar to PARP-1. The protein is localized in the nucleus in vivo and may account for the residual poly(ADP-ribose) synthesis observed in PARP-1-deficient cells, treated with alkylating agents or hydrogen peroxide.     

A steroid/thyroid hormone receptor superfamily member in Drosophila melanogaster that shares extensive sequence similarity with a mammalian homologue

A gene in Drosophila melanogaster that maps cytologically to 2C1-3 on the distal portion of the X-chromosome encodes a member of the steroid/thyroid hormone receptor superfamily. The gene was isolated from an embryonic cDNA library using an oligonucleotide probe that specifies the consensus amino acid sequence in the DNA-binding domain of several human receptors. The conceptual amino acid sequence of 2C reveals at least four regions of homology that are shared with all identified vertebrate receptors. Region I includes the two cysteine-cysteine zinc fingers that comprise a DNA-binding domain which typifies all members of the superfamily. In addition, three regions (Regions II-IV) in the carboxy-terminal portion of the protein that encode the putative hormone-binding domain of the 2C gene product resemble similar sequences in vertebrate steroid/thyroid hormone receptors. The similarity suggests that this Drosophila receptor possesses many of the regulatory functions attributed to these regions in vertebrate counterparts. A portion of Region II also resembles part of the human c-jun oncoprotein's leucine zipper, which in turn, has been demonstrated to be the heterodimerization site between the jun and fos oncoproteins. The 2C receptor-like protein most resembles the mouse H2RII binding protein, a member of the superfamily which has been implicated in the regulation of major histocompatibility complex (MHC) class I gene expression. These two gene products are 83% identical in the DNA-binding domain and 50% identical in the putative hormone-binding domain, although no ligand has been identified for either protein. The high degree of similarity in the hormone-binding domain between the 2C protein and the H2RII binding protein outside regions II-IV suggests specific functional roles which are not shared by other members of the superfamily.     

Cloning, expression and chromosomal localization of human Ca2+/calmodulin-dependent protein kinase kinase

A human cDNA clone encoding the calcium/calmodulin-dependent protein kinase kinase (CaMKK) was isolated by RT-PCR amplification of the fragment corresponding to the conserved kinase catalytic domain followed by rapid amplification of cDNA ends and cDNA library screening. Compilation of nucleotide sequencing data yielded a consensus cDNA sequence of 1.9 kb with an open reading frame of 1,251 nucleotides in length which translates to a polypeptide of 417 amino acids (47 kd). It showed significant homology to the rat brain CaMKK isozymes. The human CaMKK, which was expressed as a Flag-tagged protein in human non-small cell lung cancer H-1299 cells followed by immunoprecipitation with anti-Flag antibody, was shown to phosphorylate recombinant human CaMK I in a calcium/CaM-dependent fashion. Northern blot analysis revealed that human CaMKK is ubiquitously expressed, with brain showing the highest level of expression. The CaMKK gene is localized to human chromosome 12. The presence of cDNA clones with divergent 3' terminal sequences suggests a family of CaMKK variants which may arise from alternative splicing.     

Drosophila RNA polymerase II elongation factor DmS-II has homology to mouse S-II and sequence similarity to yeast PPR2.

DmSII is a Drosophila RNA polymerase II elongation factor which suppresses pausing by RNA polymerase II at specific sites on double stranded templates. Using antibodies produced against the purified protein, a Drosophila cDNA expression library was screened and a cDNA was isolated which encoded a portion of DmSII. When this cDNA was used to probe Kc cell mRNA the predominant species was found to be 1.4 kb in length. The original cDNA was used to screen a Drosophila Kc cell cDNA library resulting in the isolation of a 1.4 kb cDNA which was then sequenced. The deduced protein sequence for DmSII exhibited high similarity to mouse SII protein sequence. In addition, significant sequence similarity was found with the protein encoded by the yeast gene PPR2, which is involved in regulation of URA4 gene expression. The comparison of amino acid sequences suggests that DmSII is comprised of two domains homologous to mouse SII separated by a flexible, serine rich region of low homology. The shorter yeast protein has sequence similarity only to the carboxy terminal domain.     

A lipase isolated from the silkworm Bombyx mori shows antiviral activity against nucleopolyhedrovirus

A protein showing strong antiviral activity against Bombyx mori nucleopolyhedrovirus (BmNPV) was purified from the digestive juice of B. mori larvae. A homology search of the deduced amino acid sequence of the protein cDNA revealed 56% homology with Drosophila melanogaster lipase and 21% homology with human lipase. As lipase activity of the protein was confirmed in vitro, this protein was designated Bmlipase-1. Northern blot analysis showed that the Bmlipase-1 gene is expressed in the midgut but not in other tissues, nor is it activated by BmNPV infection. In addition, the Bmlipase-1 gene was shown not to be expressed in the molting and wandering stages, indicating that the gene is hormonally regulated. Our results suggest that an insect digestive enzyme has potential as a physiological barrier against BmNPV at the initial site of viral infection.     

Structure and characteristics of an endo-beta-1,4-glucanase, isolated from Trametes hirsuta, with high degradation to crystalline cellulose

Trametes hirsuta produced cellulose-degrading enzymes when it was grown in a cellulosic medium such as Avicel or wheat bran. An endo-beta-1,4-glucanase (ThEG) was purified from the culture filtrate, and the gene and the cDNA were isolated. The gene consisted of an open reading frame encoding 384 amino acids, interrupted by 11 introns. The whole sequence showed high homology with that of family 5 glycoside hydrolase. The properties of the recombinant enzyme (rEG) in Aspergillus oryzae were compared with those of the En-1 from Irpex lacteus, which showed the highest homology among all the endoglucanases reported. The rEG activity against Avicel was about 8 times higher than that of En-1 when based on CMC degradation. A remarkable structural difference between the two enzymes was the length of the linker connecting the cellulose-binding domain to the catalytic domain.     

The rat interleukin 10 receptor: cloning and sequencing of cDNA coding for the alpha-chain protein sequence, and demonstration by western blotting of expression in the rat brain

cDNA coding for the alpha chain of the rat interleukin 10 (IL-10) receptor was amplified by polymerase chain reaction (PCR), cloned and sequenced. The nucleic acid coding sequence exhibited 88% and 68% homology with the mouse and human IL-10 receptor sequences, respectively. The translated protein exhibited 83% and 61% homology with the mouse and human IL-10 receptor proteins. Specific antibodies were raised to the extracellular domain of the rat IL-10 receptor expressed as a secreted protein in recombinant Drosophila S2 cells. Western blotting using these antibodies demonstrated the presence of the IL-10 receptor in five major regions of the rat brain (cortex, cerebellum, hippocampus, hypothalamus and pituitary), supporting a role for IL-10 as a central regulator of inflammation.