Molecular characterization, tissue expression, and mapping of a novel Siglec-like gene (SLG2) with three splice variants.

The sialic acid binding immunglobulin-like lectin (Siglec) family is a recently described member of the immunoglobulin superfamily. Within the Siglec family, there exists a subgroup, which bears a high degree of homology with the molecule CD33 (Siglec-3), and has thus been designated the CD33-like subgroup of Siglecs. Members of this subgroup have been localized to chromosome 19q13.4. Through the positional candidate approach, we identified a novel potential member of this subgroup of Siglecs. We have characterized the complete genomic structure of this gene, determined its chromosomal localization, its homology to other members of the Siglec family, and its tissue expression profile. This new Siglec-like gene is comprised of 11 exons, with 10 intervening introns, and is localized 278 kb telomeric to Siglec-9 and 35 kb centromeric to Siglec-8 and on chromosome 19q13.4. The coding region consists of 2094 base pairs, and encodes for a putative 76.6 kDa protein. All Siglec-conserved structural features, including V-set domain, three C-set domains, transmembrane domain, ITIM and SLAM motifs, were found in this Siglec-like gene. Also, it has the conserved amino acids essential for sialic acid binding. The Siglec-like gene has 40-66% homology with members of the CD33-like subgroup, including Siglecs 5-9. Through RT-PCR we have examined the expression profile of this new gene in a panel of human tissues and found it to be primarily expressed in the bone marrow, spleen, brain, small intestine, colon, and spinal cord. We were also able to identify three different splice variants of the new gene. This gene may represent the latest novel member of the CD33-like subgroup of Siglecs, and, given its high degree of homology, it may also serve a regulatory role in the proliferation and survival of a particular hematopoietic stem cell lineage, as has been found for CD33 and Siglec-7.     

The Unique Envelope Gene of the Subgroup J Avian Leukosis Virus Derives from ev/J Proviruses, a Novel Family of Avian Endogenous Viruses

A new subgroup of avian leukosis virus (ALV), designated subgroup J, was identified recently. Viruses of this subgroup do not cross-interfere with viruses of the avian A, B, C, D, and E subgroups, are not neutralized by antisera raised against the other virus subgroups, and have a broader host range than the A to E subgroups. Sequence comparisons reveal that while the subgroup J envelope gene includes some regions that are related to those found in env genes of the A to E subgroups, the majority of the subgroup J gene is composed of sequences either that are more similar to those of a member (E51) of the ancient endogenous avian virus (EAV) family of proviruses or that appear unique to subgroup J viruses. These data led to the suggestion that the ALV-J env gene might have arisen by multiple recombination events between one or more endogenous and exogenous viruses. We initiated studies to investigate the origin of the subgroup J envelope gene and in particular to determine the identity of endogenous sequences that may have contributed to its generation. Here we report the identification of a novel family of avian endogenous viruses that include env coding sequences that are over 95% identical to both the gp85 and gp37 coding regions of subgroup J viruses. We call these viruses the ev/J family. We also report the isolation of ev/J-encoded cDNAs, indicating that at least some members of this family are expressed. These data support the hypothesis that the subgroup J envelope gene was acquired by recombination with expressed endogenous sequences and are consistent with acquisition of this gene by only one recombination event.     

Cloning and molecular characterization of two splice variants of a new putative member of the Siglec-3-like subgroup of Siglecs.

The sialic acid binding immunoglobulin-like lectin (Siglec) family is a recently described member of the immunoglobulin superfamily. Within this Siglec family there exists a subgroup of molecules which bear a very high degree of homology with the molecule Siglec-3 (CD33), and has thus been designated the Siglec-3-like subgroup of Siglecs. The members of this subgroup have been localized to chromosome 19q13.4, through both in situ hybridization and precise genomic mapping at the nucleotide level. Through the positional cloning approach we have identified and characterized a Siglec-like gene (SLG), a putative novel member of the Siglec-3-like subgroup of Siglecs. We have characterized the complete genomic structure of SLG, as well as two alternative splice variants, and determined its chromosomal localization. The short isoform, SLG-S, consists of seven exons, with six intervening introns, while the longer isoform, SLG-L, consists of eight exons and seven intervening introns. The SLG gene is localized 32.9 kb downstream of Siglec-8 on chromosome 19q13.4. The putative SLG-S and SLG-L proteins, of 477 and 595 amino acid residues, respectively, show extensive homology to many members of the Siglec-3-like subgroup. This high degree of homology is conserved in the extracellular Ig-like domains, as well as in the cytoplasmic tyrosine-based motifs. Interestingly, the SLG-L protein contains two N-terminal V-set Ig-like domains, as opposed to SLG-S and other Siglec-3-like subgroup members which contain only one such domain. Through RT-PCR we have examined the expression profile of both SLG splice variants in a panel of human tissues and have found that SLG-S is highly expressed in spleen, small intestine and adrenal gland, while SLG-L exhibits high levels of expression in spleen, small intestine, and bone marrow. This gene is quite likely the latest novel member of the CD33-like subgroup of Siglecs, and given its high degree of homology, it may also serve a regulatory role in the proliferation and survival of a particular hematopoietic stem cell lineage, as has been found for CD33 and Siglec7.     

Protocadherin family: diversity, structure, and function

Protocadherins are predominantly expressed in the nervous system, and constitute the largest subgroup within the cadherin superfamily. The recent structural elucidation of the amino-terminal cadherin domain in an archetypal protocadherin revealed unique and remarkable features: the lack of an interface for homophilic adhesiveness found in classical cadherins, and the presence of loop structures specific to the protocadherin family. The unique features of protocadherins extend to their genomic organization. Recent findings have revealed unexpected allelic and combinatorial gene regulation for clustered protocadherins, a major subgroup in the protocadherin family. The unique structural repertoire and unusual gene regulation of the protocadherin family may provide the molecular basis for the extraordinary diversity of the nervous system.     

CLMP, a novel member of the CTX family and a new component of epithelial tight junctions

The CTX family is a growing group of type I transmembrane proteins within the immunoglobulin superfamily (IgSF). They localize to junctional complexes between endothelial and epithelial cells and seem to participate in cell-cell adhesion and transmigration of leukocytes. Here, we report the identification of a new member of the CTX family. This protein, which was designated CLMP (coxsackie- and adenovirus receptor-like membrane protein), is composed of 373 amino acids including an extracellular part containing a V- and a C2-type domain, a transmembrane region and a cytoplasmic tail. CLMP mRNA was detected in a variety of both human and mouse tissues and cell lines. The protein migrated with an Mr of around 48 on SDS-PAGE and was predominantly expressed in epithelial cells within different tissues. In cultured epithelial cells, CLMP was detected in areas of cell-cell contacts. When exogenously expressed in polarized MDCK cells, CLMP was restricted to the subapical area of the lateral cell surface, where it co-localized with the tight junction markers ZO-1 and occludin. Also endogenous CLMP showed association with tight junctions, as analyzed in polarized human CACO-2 cells. This suggested a role for CLMP in cell-cell adhesion and indeed, overexpressed CLMP induced aggregation of non-polarized CHO cells. Furthermore, CLMP-expressing MDCK cells showed significantly increased transepithelial resistance, indicating a role for CLMP in junctional barrier function. Thus, we conclude that CLMP is a novel cell-cell adhesion molecule and a new component of epithelial tight junctions. We also suggest, based on phylogenetic studies, that CLMP, CAR, ESAM, and BT-IgSF form a new group of proteins within the CTX family.     

Identification of a novel aquaporin, AQP12, expressed in pancreatic acinar cells

Members of the aquaporin (AQP) water channel family are widely distributed in various tissues and contribute to the water permeability of epithelial and endothelial cells. Currently 11 members of the AQP family (AQP0-10) have been reported in mammals. Here we report the identification of AQP12, which we found by performing a BLAST program search. Northern blot analysis revealed that AQP12 was specifically expressed in the pancreas. Further analysis by in situ hybridization and RT-PCR studies showed that AQP12 was selectively localized in the acinar cells of the pancreas. To analyze the cellular localization and function of AQP12, we expressed AQP12 in Xenopus oocytes and cultured mammalian cells. Immunocytochemistry revealed that AQP12 was not targeted to the plasma membrane. The selective localization of AQP12 in pancreatic acinar cells and possibly in the intracellular organelles suggests a role of AQP12 in digestive enzyme secretion such as maturation and exocytosis of secretory granules.     

Identification of three novel proteins (SGSM1, 2, 3) which modulate small G protein (RAP and RAB)-mediated signaling pathway

We report a novel protein family consisting of three members, each of which contains RUN and TBC motifs and appears to be associated with small G protein-mediated signal transduction pathway. We named these proteins as small G protein signaling modulators (SGSM1/2/3). Northern blot analysis revealed that human SGSM2/3 are expressed ubiquitously in various tissues, whereas SGSM1 is expressed mainly in brain, heart, and testis. Mouse possessed the same protein family genes, and the in situ hybridization and immunohistochemical staining of tissue sections revealed that mouse Sgsm1/2/3 are expressed in the neurons of central nervous system, indicating the strong association of Sgsm family with neuronal function. Furthermore, endogenous Sgsm1 protein was localized in the trans-Golgi network of mouse Neuro2a cells by immunofluorescence microscopy. Expression of various cDNA constructs followed by immunoprecipitation assay revealed that human SGSM1/2/3 proteins are coprecipitated with RAP and RAB subfamily members of the small G protein superfamily. Based on these results, we postulated that the SGSM family members function as modulators of the small G protein RAP and RAB-mediated neuronal signal transduction and vesicular transportation pathways.     

KHDC1A, a Novel Translational Repressor, Induces Endoplasmic Reticulum-Dependent Apoptosis

RNA binding proteins are characterized as a new family of apoptosis inducers; however, the mechanism by which they induce apoptosis is poorly understood. KHDC1 family members were recently identified as K-homology (KH)-domain containing RNA binding proteins that are unique to eutherian mammals and highly expressed in oocytes. In this study, we report that the expression of KHDC1A induces caspase-3 dependent apoptosis and inhibits mRNA translation, and the translational repression is independent of apoptosis. We demonstrate that both the N-terminus and C-terminus of KHDC1A are required for its pro-apoptotic and translational repression activities. Furthermore, in the C-terminus of KHDC1A, a putative trans-membrane motif (TMM) is critical for these activities. In addition, the ectopically expressed KHDC1A is localized to the endoplasmic reticulum (ER) and changes the morphology of the ER. The inhibition of ER-specific caspase-12 successfully rescues KHDC1A-induced apoptosis, but not Fas-induced apoptosis. Taken together, we conclude that KHDC1A functions as a global translational repressor and induces apoptosis through an ER-dependent signaling pathway.     

Mouse and Human Neuronal Pentraxin 1 (NPTX1): Conservation, Genomic Structure, and Chromosomal Localization

We have previously identified novel members of the pentraxin family (neuronal pentraxin 1 and 2) that are expressed in the nervous system. Neuronal pentraxin 1 (NP1) was identified as a rat protein that may mediate the uptake of synaptic material and the presynaptic snake venom toxin, taipoxin. NP2 was identified as a separate gene discovered by screening for a human homolog for NP1. Here, we report human cDNA and mouse genomic DNA sequences for NP1 (gene symbol NPTX1). Human NP1 and mouse NP1 show 95 and 99% amino acid identity, respectively, with rat NP1 and conserve all potential glycosylation sites. Like rat NP1, human NP1 message is large (6.5 kb) and is exclusively localized to the nervous system. The mouse NP1 gene is 13 kb in length and contains four introns that break the coding sequence of NP1 in the same positions as the introns of the human NP2 gene. The human and mouse NP1 genes are localized to chromosome 17q25.1–q25.2 and chromosome 11e2–e1.3, respectively. These data demonstrate the existence of a separate family of pentraxin proteins that are expressed in the human brain and other tissues and that may play important roles in the uptake of extracellular material.     

Doom, a product of the Drosophila mod(mdg4) gene, induces apoptosis and binds to baculovirus inhibitor-of-apoptosis proteins.

A family of baculovirus inhibitor-of-apoptosis (IAP) genes is present in mammals, insects, and baculoviruses, but the mechanism by which they block apoptosis is unknown. We have identified a protein encoded by the Drosophila mod(mdg4) gene which bound to the baculovirus IAPs. This protein induced rapid apoptosis in insect cells, and consequently we have named it Doom. Baculovirus IAPs and P35, an inhibitor of aspartate-specific cysteine proteases, blocked Doom-induced apoptosis. The carboxyl terminus encoded by the 3' exon of the doom cDNA, which distinguishes it from other mod(mdg4) cDNAs, was responsible for induction of apoptosis and engagement of the IAPs. Doom localized to the nucleus, while the IAPs localized to the cytoplasm, but when expressed together, Doom and the IAPs both localized in the nucleus. Thus, IAPs might block apoptosis by interacting with and modifying the behavior of Doom-like proteins that reside in cellular apoptotic pathways.     

Analysis of a 1-Mb BAC contig overlapping the mouse Nkrp1 cluster of genes: cloning of three new Nkrp1 members, Nkrp1d, Nkrp1e, and Nkrp1f

The murine Nkrp1 gene family encodes three previously identified activation and inhibitory receptors expressed on natural killer (NK) cells. This family includes the gene for NKR-P1C (NK1.1), the most specific serologic marker on C57BL/6-derived NK cells and is localized in a gene cluster in the NK gene complex (NKC). To further analyze the Nkrp1 family, we constructed and analyzed a bacterial artificial chromosome contig. A genomic organization of the Nkrp1 family was obtained and three new Nkrp1 genes were isolated from interleukin-2-activated NK cells. Thus, the Nkrp1 family adds to the repertoire of receptors expressed by NK cells.     

Carcinoembryonic antigen gene family members in submandibular salivary gland: demonstration of pregnancy-specific glycoproteins by cDNA cloning

We have recently shown that human submandibular salivary gland and saliva contain a number of glycoproteins belonging to the carcinoembryonic antigen (CEA) gene family. The members of the CEA family can be divided into the CEA subgroup and the pregnancy specific beta 1 glycoprotein (PSG) subgroup. The latter glycoproteins are abundant in placenta and fetal liver. Here we report that PSG's are expressed in normal adult submandibular salivary gland. Thus, cDNA cloning and sequencing gave two clones (SG5 and SG9) which coded for glycoproteins with a domain arrangement of N-A1-A2-B2-C and a third clone (SG8) which coded for a glycoprotein with a domain arrangement of N-A1-B2-C. SG5 is identical to PSG3, and SG9 to PSG1d, while SG8 most probably corresponds to PSG2. The 3' untranslated regions of the different members of the PSG subgroup contain highly homologous segments, suggesting a common evolutionary origin.