Characterization of two novel lipocalins expressed in the Drosophila embryonic nervous system

We have found two novel lipocalins in the fruit fly Drosophila melanogaster that are homologous to the grasshopper Lazarillo, a singular lipocalin within this protein family which functions in axon guidance during nervous system development. Sequence analysis suggests that the two Drosophila proteins are secreted and possess peptide regions unique in the lipocalin family. The mRNAs of DNLaz (for Drosophila neural Lazarillo) and DGLaz (for Drosophila glial Lazarillo) are expressed with different temporal patterns during embryogenesis. They show low levels of larval expression and are highly expressed in pupa and adult flies. DNLaz mRNA is transcribed in a subset of neurons and neuronal precursors in the embryonic CNS. DGLaz mRNA is found in a subset of glial cells of the CNS: the longitudinal glia and the medial cell body glia. Both lipocalins are also expressed outside the nervous system in the developing gut, fat body and amnioserosa. The DNLaz protein is detected in a subset of axons in the developing CNS. Treatment with a secretion blocker enhances the antibody labeling, indicating the DNLaz secreted nature. These findings make the embryonic nervous system expression of lipocalins a feature more widespread than previously thought. We propose that DNLaz and DGLaz may have a role in axonal outgrowth and pathfinding, although other putative functions are also discussed.     

A novel ubiquitously expressed alpha-latrotoxin receptor is a member of the CIRL family of G-protein-coupled receptors

Poisoning with alpha-latrotoxin, a neurotoxic protein from black widow spider venom, results in a robust increase of spontaneous synaptic transmission and subsequent degeneration of affected nerve terminals. The neurotoxic action of alpha-latrotoxin involves extracellular binding to its high affinity receptors as a first step. One of these proteins, CIRL, is a neuronal G-protein-coupled receptor implicated in the regulation of secretion. We now demonstrate that CIRL has two close homologs with a similar domain structure and high degree of overall identity. These novel receptors, which we propose to name CIRL-2 and CIRL-3, together with CIRL (CIRL-1) belong to a recently identified subfamily of large orphan receptors with structural features typical of both G-protein-coupled receptors and cell adhesion proteins. Northern blotting experiments indicate that CIRL-2 is expressed ubiquitously with highest concentrations found in placenta, kidney, spleen, ovary, heart, and lung, whereas CIRL-3 is expressed predominantly in brain similarly to CIRL-1. It appears that CIRL-2 can also bind alpha-latrotoxin, although its affinity to the toxin is about 14 times less than that of CIRL-1. When overexpressed in chromaffin cells, CIRL-2 increases their sensitivity to alpha-latrotoxin stimulation but also inhibits Ca2+-regulated secretion. Thus, CIRL-2 is a functionally competent receptor of alpha-latrotoxin. Our findings suggest that although the nervous system is the primary target of low doses of alpha-latrotoxin, cells of other tissues are also susceptible to the toxic effects of alpha-latrotoxin because of the presence of CIRL-2, a low affinity receptor of the toxin.     

The Drosophila gene tailless is expressed at the embryonic termini and is a member of the steroid receptor superfamily

The zygotically active tailless (tll) gene plays a key role in the establishment of nonmetameric domains at the anterior and posterior poles of the Drosophila embryo. We have cloned the tll gene and show that it encodes a protein with striking similarity to steroid hormone receptors in both the DNA binding "finger" and ligand binding domains. tll RNA is initially expressed in embryos in two mirror-image symmetrical domains; this pattern then quickly resolves into a pattern consistent with the mutant phenotype: a posterior cap and an anterior dorsal stripe. That the tll gene may also play a role in the nervous system is suggested by its strong expression in the forming brain and transient expression in the peripheral nervous system.     

A new member of the insulin receptor family, insulin receptor-related receptor, is expressed preferentially in the kidney.

Shier and Watt isolated human and guinea pig genomic DNA encoding a putative protein, insulin receptor-related receptor (IRR), the primary structure of which is similar to that of other members of the insulin receptor family, the insulin receptor and type-I IGF receptor (J. Biol. Chem. 264, 14605-14608 (1989)). However, the expression of the IRR gene remained unknown. In this paper, we isolated the IRR cDNA from the rat brain and examined the expression of the IRR mRNA in a variety of rat tissues, including the brain, heart, lung, liver, small intestine, kidney, thymus, spleen, muscle, adipose tissue and cartilage by polymerase chain reaction. In contrast to the wide distribution of the insulin receptor and type-I IGF receptor mRNAs, the IRR mRNA is expressed preferentially in the kidney, which indicates that IRR has unique functions as a member of the insulin receptor family.     

A peritrophin-like protein expressed in the embryonic tracheae of Drosophila melanogaster.

We have cloned and sequenced a cDNA from Drosophila melanogaster that encodes a protein homologous to the peritrophins, a family of chitin-binding proteins from the peritrophic matrix of insects. Unexpectedly, the gene, Gasp, is expressed in the embryonic tracheae. We suggest that this family of proteins may be present in other tissues than the peritrophic matrix, particularly where nutrient or gas exchange are important, and/or where invasion by parasites or viruses is possible. We have also mapped two similar genes that had been sequenced by the Berkeley Drosophila Genome Project, and find that these three very similar genes are not clustered, but are located on three different chromosomes.     

Eos: a novel member of the Ikaros gene family expressed predominantly in the developing nervous system

We identified a novel member of the Ikaros gene family, which has critical roles in the development of lymphoid lineages. This gene, which we named Eos, was expressed predominantly in the developing central and peripheral nervous system. Eos protein could interact with itself and Ikaros protein through its C-terminal portion in the yeast two hybrid assay. These findings suggested that Eos may have important roles in neural development similarly to the Ikaros family in the development of hemolymphoid tissue.     

Try29F,a new member of the Drosophila trypsin-like protease gene family,is specifically expressed in the posterior embryonic midgut

The Drosophila melanogaster try29F gene encodes a protein that shares all known features of serine proteases, like residues known to be involved in substrate specificity, catalysis and disulfide bond formation. In situ hybridization to mRNA in whole mount embryos shows that the expression of try29F is restricted to the posterior midgut during late embryogenesis.     

CD300 antigen like family member G: A novel Ig receptor like protein exclusively expressed on capillary endothelium

We report the characteristics of CD300LG, a member of the CD300 antigen like family. Its genomic structure is similar in both mouse and human, and at least four isoforms exist in both species. The amino acid sequence of the immunoglobulin (Ig) V like domain of CD300LG showed approximately 35% identity to those of the polymeric Ig receptor (pIgR) and Fcalpha/muR. Interestingly, mouse CD300LG proteins were uniquely expressed on capillary endothelium. Immunoelectron microscopy revealed that mouse CD300LG is localized on both apical and basolateral plasma membranes, as well as on intracellular vesicular structures, in the capillary endothelium. Transcytosis assays using polarized MDCK epithelial cells showed that CD300LG could be transcytosed bidirectionally. Furthermore, CD300LG exogenously expressed on HeLa cells could take up IgA2 and IgM, but not IgG. These results suggest that CD300LG might play an important role in molecular traffic across the capillary endothelium.     

A novel member of the integrin receptor family mediates Arg-Gly-Asp- stimulated neutrophil phagocytosis

Human neutrophils (PMN) express a heterodimeric receptor that has ligand binding specificity for the Arg-Gly-Asp (RGD) sequence within many adhesive proteins. A monoclonal antibody, B6H12, which binds to this receptor, inhibits both RGD-mediated ligand binding and stimulation of IgG-mediated phagocytosis by fibronectin, fibrinogen, vitronectin, von Willebrand's factor, and collagen type IV. By several criteria this receptor is neither a known very late antigen, a known cytoadhesin (gp IIb/IIIa-vitronectin receptor), nor a member of the LFA-1, Mac-1, p150,95 group of integrin receptors. Ligand binding via this receptor is rapidly inactivated by products of the myeloperoxidase-hydrogen peroxide-halide system of PMN. We conclude that this receptor, for which we propose the name leukocyte response integrin, is a signal-transducing molecule on PMN which may have a significant early role in modulation of PMN function at inflammatory sites.     

A new member of the prolactin-growth hormone gene family expressed in mouse placenta.

Mouse placenta has been found to contain an mRNA that encodes a previously unidentified member of the prolactin-growth hormone family. This 1.1-kb mRNA (designated PRP mRNA) was detected as a cDNA clone that hydridized to a cDNA clone of mouse proliferin, a recently described growth-associated placental protein related to prolactin. PRP mRNA levels are highest in the fetal part of the placenta and peak at day 12 of gestation, decreasing gradually until term. The 972-bp sequence of PRP mRNA, determined from two cDNA clones, encodes a protein of 244 amino acid residues that has a hydrophobic leader sequence. The protein encoded by PRP mRNA has significant homology to all of the members of the prolactin family, yet is different from each of them; it also differs from mouse placental lactogen. Nucleotide sequence homology is most extensive between PRP and proliferin mRNAs, particularly at their 5' ends, where they share 92 of the first 97 nucleotides.     

Cloning of a novel member of the low-density lipoprotein receptor family

A gene encoding a novel transmembrane protein was identified by DNA sequence analysis within the insulin-dependent diabetes mellitus (IDDM) locus IDDM4 on chromosome 11q13. Based on its chromosomal position, this gene is a candidate for conferring susceptibility to diabetes. The gene, termed low-density lipoprotein receptor related protein 5 (LRP5), encodes a protein of 1615 amino acids that contains conserved modules which are characteristic of the low-density lipoprotein (LDL) receptor family. These modules include a putative signal peptide for protein export, four epidermal growth factor (EGF) repeats with associated spacer domains, three LDL-receptor (LDLR) repeats, a single transmembrane spanning domain, and a cytoplasmic domain. The encoded protein has a unique organization of EGF and LDLR repeats; therefore, LRP5 likely represents a new category of the LDLR family. Both human and mouse LRP5 cDNAs have been isolated and the encoded mature proteins are 95% identical, indicating a high degree of evolutionary conservation.     

MDC-L, a novel metalloprotease disintegrin cysteine-rich protein family member expressed by human lymphocytes.

The metalloprotease disintegrin cysteine-rich (MDC) proteins are a recently identified family of transmembrane proteins that function in proteolytic processing of cell surface molecules and in cell adhesion. Since lymphocytes must interact with a constantly changing environment, we hypothesized that lymphocytes would express unique MDC proteins. To identify MDC proteins expressed in human lymph node, a polymerase chain reaction-based strategy combined with degenerate oligonucleotide primers was employed. We report here the identification of MDC-L (ADAM 23), a novel member of the MDC protein family. The results obtained from cDNA cloning and Northern blot analysis of mRNA isolated from various lymphoid tissues indicate that a 2.8-kilobase mRNA encoding a transmembrane form, MDC-Lm, and a 2.2-kilobase mRNA encoding a secreted form, MDC-Ls, are expressed in a tissue-specific manner. MDC-L mRNA was shown to be predominantly expressed in secondary lymphoid tissues, such as lymph node, spleen, small intestine, stomach, colon, appendix, and trachea. Furthermore, immunohistochemical staining with an anti-MDC-L antibody demonstrated that cells with typical lymphocyte morphology are responsible for expression of the MDC-L antigen in these lymphoid tissues. MDC-Lm was found to be expressed on the surface of human peripheral blood lymphocytes and transformed B- and T-lymphocyte cell lines as an 87-kDa protein. Thus, we have identified a novel lymphocyte-expressed MDC protein family member.     

Neuroglobin, a novel member of the globin family, is expressed in focal regions of the brain.

Hemoproteins are widely distributed among unicellular eukaryotes, plants, and animals. In addition to myoglobin and hemoglobin, a third hemoprotein, neuroglobin, has recently been isolated from vertebrate brain. Although the functional role of this novel member of the globin family remains unclear, neuroglobin contains a heme-binding domain and may participate in diverse processes such as oxygen transport, oxygen storage, nitric oxide detoxification, or modulation of terminal oxidase activity. In this study we utilized in situ hybridization (ISH) and RT-PCR analyses to examine the expression of neuroglobin in the normoxic and hypoxic murine brain. In the normoxic adult mouse, neuroglobin expression was observed in focal regions of the brain, including the lateral tegmental nuclei, the preoptic nucleus, amygdala, locus coeruleus, and nucleus of the solitary tract. Using ISH and RT-PCR techniques, no significant changes in neuroglobin expression in the adult murine brain was observed in response to chronic 10% oxygen. These results support the hypothesis that neuroglobin is a hemoprotein that is expressed in the brain and may have diverse functional roles.     

A novel member of the subtilisin-like protease family from Streptomyces albogriseolus.

We previously isolated three extracellular endogenous enzymes from a Streptomyces albogriseolus mutant strain which were targets of Streptomyces subtilisin inhibitor (SSI) (S. Taguchi, A. Odaka, Y. Watanabe, and H. Momose, Appl. Environ. Microbiol. 61:180-186, 1995). In the present study, of the three enzymes the largest one, with a molecular mass of 45 kDa (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), termed SAM-P45, has been characterized in detail. The entire gene encoding SAM-P45 was cloned as an approximately 10-kb fragment from S. albogriseolus S-3253 genomic DNA into an Escherichia coli host by using a shuttle plasmid vector. The amino acid sequence corresponding to the internal region of SAM-P45, deduced from the nucleotide sequence of the gene, revealed high homology, particularly in three regions around the active-site residues (Asp, His, and Ser), with the amino acid sequences of the mature domain of subtilisin-like serine proteases. In order to investigate the enzymatic properties of this protease, recombinant SAM-P45 was overproduced in Streptomyces coelicolor by using a strong SSI gene promoter. Sequence analysis of the SAM-P45 gene and peptide mapping of the purified SAM-P45 suggested that it is synthesized as a large precursor protein containing a large C-terminal prodomain (494 residues) in addition to an N-terminal preprodomain (23 and 172 residues). A high proportion of basic amino acids in the C-terminal prodomain was considered to serve an element interactive with the phospholipid bilayer existing in the C-terminal prodomain, as found in other membrane-anchoring proteases of gram-positive bacteria. It is noteworthy that SAM-P45 was found to prefer basic amino acids to aromatic or aliphatic amino acids in contrast to subtilisin BPN', which has a broad substrate specificity. The hydrolysis by SAM-P45 of the synthetic substrate (N-succinyl-L-Gly-L-Pro-L-Lys-p-nitroanilide) most preferred by this enzyme was inhibited by SSI, chymostatin, and EDTA. The proteolytic activity of SAM-P45 was stimulated by the divalent cations Ca2+ and Mg2+. From these findings, we conclude that SAM-P45 interacts with SSI and can be categorized as a novel member of the subtilisin-like serine protease family.     

Characterization of Sptrx, a novel member of the thioredoxin family specifically expressed in human spermatozoa

Thioredoxins (Trx) are small ubiquitous proteins that participate in different cellular processes via redox-mediated reactions. We report here the identification and characterization of a novel member of the thioredoxin family in humans, named Sptrx (sperm-specific trx), the first with a tissue-specific distribution, located exclusively in spermatozoa. Sptrx open reading frame encodes for a protein of 486 amino acids composed of two clear domains: an N-terminal domain consisting of 23 highly conserved repetitions of a 15-residue motif and a C-terminal domain typical of thioredoxins. Northern analysis and in situ hybridization shows that Sptrx mRNA is only expressed in human testis, specifically in round and elongating spermatids. Immunostaining of human testis sections identified Sptrx protein in spermatids, while immunofluorescence and immunogold electron microscopy analysis demonstrated Sptrx localization in the cytoplasmic droplet of ejaculated sperm. Sptrx appears to have a multimeric structure in native conditions and is able to reduce insulin disulfide bonds in the presence of NADPH and thioredoxin reductase. During mammalian spermiogenesis in testis seminiferous tubules and later maturation in epididymis, extensive reorganization of disulfide bonds is required to stabilize cytoskeletal sperm structures. However, the molecular mechanisms that control these processes are not known. The identification of Sptrx with an expression pattern restricted to the postmeiotic phase of spermatogenesis, when the sperm tail is organized, suggests that Sptrx might be an important factor in regulating critical steps of human spermiogenesis.