Identification and molecular characterization of a novel member of the siglec family (SIGLEC9)

Using the positional cloning approach, we have identified siglec-9 (HGMW-approved symbol SIGLEC9) a novel member of the sialic acid-binding Ig-like lectin (Siglec) family, which belongs to the immunoglobulin superfamily (IgSF). We characterized the genomic structure of this gene and determined its chromosomal localization, its homology to other members of the siglec family, and its tissue expression profile. The siglec-9 gene is composed of seven exons, with six intervening introns. The coding region consists of 1392 nucleotides and produces a 463-amino-acid protein. Furthermore, we have localized this gene to 19q13.4, 43.19 kb more telomeric than KLK14 (a member of the kallikrein gene family) through genomic sequencing data and restriction mapping with EcoRI. This novel siglec shows a high degree of homology to many members of the siglec family, including siglec-7 (80%), siglec-8 (72%), siglec-5 (65%), and CD33 (64%). This high degree of homology is also conserved in the extracellular Ig-like domains. Through RT-PCR, we have examined the expression of siglec-9 in a large number of tissues and have found relatively high-level expression in bone marrow, placenta, spleen, and fetal liver. Based on its homology to CD33, we speculate that this gene may also have some utility as a target for immunological antineoplastic therapy.     

Identification of a novel member in the family Albulidae (bonefishes)

A novel Caribbean species Albula sp. cf. vulpes in the family Albulidae (bonefishes) was diagnosed through genetic and morphometric study. Phylogenies derived from 16S rRNA sequences revealed deeply separated lineages among Caribbean bonefishes. Mitochondrial DNA sequence divergences indicated a separation between 3·0 and 5·2 million years before present (b.p.). Cytochrome b phylogenies further supported the classification of A. sp. cf. vulpes as a novel albulid. Morphological variability revealed several differences between A. sp. cf. vulpes and other Caribbean species. A microsatellite library was developed to discern hybridization rates among the species. Microsatellite analyses revealed low levels of hybridization between some members in the complex. One instance of backcrossing was found between A. vulpes×A. sp. B and a pure A. sp. B, indicating that hybrids may have reduced fitness or may be reproductively isolated due to temporal–spatial spawning habitat differences.     

Identification and characterization of rDJL, a novel member of the DnaJ protein family, in rat testis

Applying the method of segmentation of seminiferous tubules combined with DDRT-PCR and cDNA library screening, a novel DnaJ homologue, rDJL was identified in rat testis. The reading frame encodes a protein of 223 amino acid residues containing J domain in the NH2 terminal region. rDJL gene is expressed mainly in testis and rDJL protein was immunolocalized notably in the acrosome region of spermatozoa. Immunoprecipitation experiments showed that rDJL interacted with Hsc70 and clathrin protein. When CHO cells were treated with EGF, rDJL and clathrin protein were found to be colocalized and be concentrated as endosome vesicles. The present findings suggest that rDJL functions as co-chaperone to Hsc70, participates in vesicular trafficking and may play an important role in acrosomogenesis.     

Identification of a novel testis-specific member of the phosphatidylethanolamine binding protein family,pebp-2

The phosphatidylethanolamine binding proteins (pebps) are an evolutionarily conserved family of proteins recently implicated in mitogen-activated protein (MAP) kinase pathway regulation, where they are called raf kinase inhibitory proteins. Here, we describe the cloning, cellular localization, and partial characterization of a new member, pebp-2, with potential roles in male fertility. Expression data show that pebp-2 is a testis-specific 21-kDa protein found within late meiotic and haploid germ cells in a stage-specific pattern that is temporally distinct from that of pebp-1. Sequence analyses suggest that pebp-2 forms a distinct subset of the pebp family within mammals. Database analyses revealed the existence of a third subset. Analysis suggests that the specificity/regulation of the distinct pebps subsets is likely to be determined by the amino terminal 40 amino acids or the 3' untranslated region, where the majority of sequence differences occur. Protein homology modeling suggests that pebp-2 protein is, however, topologically similar to other pebps and composed of Greek key fold motifs, a dominant beta-sheet formed from five anti-parallel beta strands forming a shallow groove associated with a putative phosphatidylethanolamine binding site. The pebp-2 gene is intronless and data suggest that it is a retrogene derived from pebp-1. Further, pebp-2 colocalizes with members of the MAP kinase pathway in late spermatocytes and spermatids and on the midpiece of epididymal sperm. These data raise the possibility that pebp-2 is a novel participant in the MAP kinase signaling pathway, with a role in spermatogenesis or posttesticular sperm maturation.     

Cloning and characterization of IL-1HY2, a novel interleukin-1 family member

The interleukin-1 (IL-1) family members play an important role in the process of inflammation and host defense. We describe here the identification and characterization of a novel member of the IL-1 family, IL-1HY2. The human IL-1HY2 protein shares significant amino acid sequence similarity (37%) with the IL-1 receptor antagonist and has a predicted three-dimensional structure similar to that of the IL-1 receptor antagonist. The IL-1HY2 gene is located in close proximity to other IL-1 family genes on human chromosome 2, and the genomic organization of the IL-1HY2 gene is highly conserved with other IL-1 family members. IL-1HY2 protein is secreted from mammalian cells, and the purified recombinant IL-1HY2 protein binds soluble IL-1 receptor type I. IL-1HY2 is expressed in human skin, spleen, and tonsil. Immunohistochemical analysis showed that the IL-1HY2 protein is expressed in the basal epithelia of skin and in proliferating B cells of the tonsil. These data suggest that IL-1HY2 is a novel IL-1 family member and that it may participate in a network of IL-1 family members to regulate adapted and innate immune responses.     

Capitella sp. I homeobrain-like, the first lophotrochozoan member of a novel paired-like homeobox gene family

The paired-like class of homeobox genes contains numerous distinct families, many of which have been implicated in a variety of developmental functions. We report the isolation and expression of a gene with high similarity to Drosophila melanogaster homeobrain from the polychaete annelid Capitella sp. I. The homeobrain-like (hbnl) gene is a paired-like gene that contains a conserved homeodomain, octapeptide region, alanine stretches, and an OAR domain. Gene orthology analyses of the homeodomain from CapI-hbnl places this gene in a new family of paired-like homeodomain genes that includes D. melanogaster homeobrain (hbn) and representatives from all major bilaterian clades as well as a cnidarian gene. CapI-hbnl expression is largely restricted to subsets of cells in the brain and eyes during larval development in Capitella sp. I. The earliest expression of CapI-hbnl is in small discrete cell clusters in the cerebral ganglia. This expression persists through late larval developmental stages whereas expression is absent in postmetamorphic juveniles. Outside the brain, expression is present on the ventral side of the larva in two small cell clusters, at the brain/pharyngeal border and in the anterior-most segment. CapI-hbnl shares features of brain expression with hbn, although in contrast to hbn, which is expressed along the length of the ventral nerve cord, CapI-hbnl has a restricted anterior expression pattern. CapI-hbnl represents an important neural marker for characterization of the annelid nervous system.     

The chicken NKX2.8 homeobox gene: A novel member of the NK-2 gene family

 We have isolated the chicken homeobox gene NKX2.8, which represents a novel member of the NK-2 gene family. Besides the homeodomain, the NKX2.8 protein contains two other conserved sequences, a TN and an NK2 domain. NKX2.8 is expressed in the ventral foregut, the developing heart, in the epithelial layers of the branchial arches and in the dorsal mesocardium. Thus, its expression overlaps partially, but also differs significantly from another chicken tinman orthologue, the NKX2.5 gene. It is suggestive that NKX2.8 and NKX2.5 play a cooperative role in early heart development. Received: 28 January 1997 / Accepted: 8 February 1997     

Bcl-G, a novel pro-apoptotic member of the Bcl-2 family

A new member of the Bcl-2 family was identified, Bcl-G. The human BCL-G gene consists of 6 exons, resides on chromosome 12p12, and encodes two proteins through alternative mRNA splicing, Bcl-G(L) (long) and Bcl-G(S) (short) consisting of 327 and 252 amino acids in length, respectively. Bcl-G(L) and Bcl-G(S) have identical sequences for the first 226 amino acids but diverge thereafter. Among the Bcl-2 homology (BH) domains previously recognized in Bcl-2 family proteins, the BH3 domain is found in both Bcl-G(L) and Bcl-G(S), but only the longer Bcl-G(L) protein possesses a BH2 domain. Bcl-G(L) mRNA is expressed widely in adult human tissues, whereas Bcl-G(S) mRNA was found only in testis. Overexpression of Bcl-G(L) or Bcl-G(S) in cells induced apoptosis although Bcl-G(S) was far more potent than Bcl-G(L). Apoptosis induction by Bcl-G(S) depended on the BH3 domain and was suppressed by coexpression of anti-apoptotic Bcl-X(L) protein. Bcl-X(L) also coimmunoprecipitated with Bcl-G(S) but not with mutants of Bcl-G(S) in which the BH3 domain was deleted or mutated or with Bcl-G(L). Bcl-G(S) was predominantly localized to cytosolic organelles, whereas Bcl-G(L) was diffusely distributed throughout the cytosol. A mutant of Bcl-G(L) in which the BH2 domain was deleted displayed increased apoptotic activity and coimmunoprecipitated with Bcl-X(L), suggesting that the BH2 domain autorepresses Bcl-G(L).     

SID1 transmembrane family, member 2 (Sidt2): a novel lysosomal membrane protein

In a recent proteomic study of lysosomal proteins [10], we identified SID1 transmembrane family, member 2 (Sidt2) as a novel lysosomal membrane protein candidate. The Sidt2 gene encodes an 832-amino acid residues protein with a calculated molecular mass of 94.5 kDa. Bioinformatic analysis showed that Sidt2 is a multipass transmembrane protein that contains 10 putative N-glycosylation sites (NxS/T) and two potential tyrosine-based sorting signals (YGSF and YDTL). Using specific anti-Sidt2 antibody and lysosomal markers, the lysosomal localization of Sidt2 was determined by immunofluorescence. Furthermore, using subcellular fractionation techniques, we demonstrated that Sidt2 is a lysosomal integral membrane protein. Endogenous Sidt2 was detected in multiple tissues of mouse and rat with approximately 120-130 kDa molecular weights due to extensive glycosylation. After digestion with PNGase F, the apparent molecular mass of Sidt2 decreased to the predicted value of 95 kDa. In rats, Sidt2 was highly expressed in the liver, brain, and kidney, whereas no or little expression was found in the skeletal muscles, heart, and other tissues. In summary, Sidt2 is a highly glycosylated lysosomal integral membrane protein that shows tissue-specific expression.     

Identification and characterization of a novel member of olfactomedin-related protein family, hGC-1, expressed during myeloid lineage development

We have cloned a novel hematopoietic granulocyte colony-stimulating factor (G-CSF)-induced olfactomedin-related glycoprotein, termed hGC-1 (human G-CSF-stimulated clone-1). mRNA differential display was used in conjunction with a modified two-phase liquid culture system. Cultures were enriched for early precursors of erythroid, myeloid, and megakaryocytic lineages, which were isolated after induction with erythropoietin, G-CSF, and thrombopoietin, respectively. RNA from the enriched cells was subjected to differential display analysis to identify lineage-specific expressed genes. One clone specifically induced by G-CSF, hGC-1, was characterized. The 2861 bp cDNA clone of hGC-1 contained an open reading frame of 1530 nucleotides, translating into a protein of 510 amino acids with a signal peptide and six N-linked glycosylation motifs. The protein sequence of hGC-1 showed it to be a glycoprotein of the olfactomedin family, which includes olfactomedin, TIGR, Noelin-2 and latrophilin-1. Olfactomedin-like genes show characteristic tissue-restricted patterns of expression; the specific tissues expressing these genes differ among the family members. hGC-1 was strongly expressed in the prostate, small intestine, and colon, moderately expressed in the bone marrow and stomach, and not detectable in other tissues. In vitro translation and ex vivo expression showed hGC-1 to be an N-linked glycoprotein. The hGC-1 gene locus mapped to chromosome 13q14.3. Together, our findings indicate that hGC-1 is primarily expressed as an extracellular olfactomedin-related glycoprotein during normal myeloid-specific lineage differentiation, suggesting the possibility of a matrix-related function for hGC-1 in differentiation.