Putative Membrane-spanning Domains Research Articles

Elovl4a participates in LC-PUFA biosynthesis and is regulated by PPAR\u03b1\u03b2 in golden pompano Trachinotus ovatus (Linnaeus 1758)

The elongases of very long-chain fatty acids (Elovls) are responsible for the rate-limiting elongation process in long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis. The transcription factor, PPARα, regulates lipid metabolism in mammals; however, the detailed mechanism whereby PPARαb regulates Elovls remains largely unknown in fish. In the present study, we report the full length cDNA sequence of Trachinotus ovatus Elovl4a (ToElovl4a), which encodes a 320 amino acid polypeptide that possesses five putative membrane-spanning domains, a conserved HXXHH histidine motif and an ER retrieval signal. Phylogenetic analysis revealed that the deduced protein of ToElovl4a is highly conserved with the Oreochromis niloticus corresponding homologue. Moreover, functional characterization by heterologous expression in yeast indicated that ToElovl4a can elongate C18 up to C20 polyunsaturated fatty acids. A nutritional study showed that the protein expressions of ToElovl4a in the brain and liver were not significantly affected among the different treatments. The region from PGL3-basic-Elovl4a-5 (−148 bp to +258 bp) is defined as the core promoter via a progressive deletion mutation of ToElovl4a. The results from promoter activity assays suggest that ToElovl4a transcription is positively regulated by PPARαb. Mutation analyses indicated that the M2 binding site of PPARαb is functionally important for protein binding, and transcriptional activity of the ToElovl4a promoter significantly decreased after targeted mutation. Furthermore, PPARαb RNA interference reduced ToPPARαb and ToElovl4a expression at the protein levels in a time-dependent manner. In summary, PPARαb may promote the biosynthesis of LC-PUFA by regulating ToElovl4a expression in fish.

Characterization and expression profiles of aquaporins (AQPs) 1a and 3a in mud loach Misgurnus mizolepis after experimental challenges

Two distinct cDNAs encoding aquaporins (mmAQPs 1a and 3a) were isolated and characterized from mud loach Misgurnus mizolepis. The identified mud loach AQP cDNAs encode for polypeptides of 260 and 302 amino acids. Topology predictions confirmed six putative membrane-spanning domains connected by five loops and the N- and C-terminal domains being cytoplasmic. The mud loach AQPs 1a and 3a showed broad distribution in multiple tissues including immune-responsive tissues as well as osmoregulatory tissues. Hence, the diversity of AQP distribution and expression possibly indicated its differential functions in the regulation of fluid movement in response to environmental stimuli. The transcription of mmAQP genes was differentially modulated by immune challenges. In particular, the mmAQP3a expression level in the liver was more responsive to immune challenges than that of mmAQP1a. Taken together, fish stimulation or infection resulted in significant modulation of mud loach AQP genes, suggesting potential functional roles of these proteins in piscine pathophysiological process.

Molecular cloning and functional characterization of a glucose transporter (CsGLUT) in Clonorchis sinensis.

A complementary DNA (cDNA) encoding a glucose transporter of Clonorchis sinensis (CsGLUT) was isolated from the adult C. sinensis cDNA library. The open reading frame of CsGLUT cDNA consists of 1653 base pairs that encode a 550-amino acid residue protein. Hydropathy analysis suggested that CsGLUT possess 12 putative membrane-spanning domains. The Northern blot analysis result using poly(A)(+)RNA showed a strong band at ~2.1 kb for CsGLUT. When expressed in Xenopus oocytes, CsGLUT mediated the transport of radiolabeled deoxy-D-glucose in a time-dependent but sodium-independent manner. Concentration-dependency results showed saturable kinetics and followed the Michaelis-Menten equation. Nonlinear regression analyses yielded a Km value of 588.5 ± 53.0 μM and a Vmax value of 1500.0 ± 67.5 pmol/oocyte/30 min for [1,2-(3)H]2-deoxy-D-glucose. No trans-uptakes of bile acid (taurocholic acid), amino acids (tryptophan and arginine), or p-aminohippuric acid were observed. CsGLUT-mediated transport of deoxyglucose was significantly and concentration-dependently inhibited by radio-unlabeled deoxyglucose and D-glucose. 3-O-Methylglucose at 10 and 100 μM inhibited deoxyglucose uptake by ~50 % without concentration dependence. No inhibitory effects by galactose, mannose, and fructose were observed. This work may contribute to the molecular biological study of carbohydrate metabolism and new drug development of C. sinensis.

Reduced sodium/proton exchanger NHE3 activity causes congenital sodium diarrhea.

Congenital sodium diarrhea (CSD) refers to an intractable diarrhea of intrauterine onset with high fecal sodium loss. CSD is clinically and genetically heterogeneous. Syndromic CSD is caused by SPINT2 mutations. While we recently described four cases of the non-syndromic form of CSD that were caused by dominant activating mutations in intestinal receptor guanylate cyclase C (GC-C), the genetic cause for the majority of CSD is still unknown. Therefore, we aimed to determine the genetic cause for non-GC-C non-syndromic CSD in 18 patients from 16 unrelated families applying whole-exome sequencing and/or chromosomal microarray analyses and/or direct Sanger sequencing. SLC9A3 missense, splicing and truncation mutations, including an instance of uniparental disomy, and whole-gene deletion were identified in nine patients from eight families with CSD. Two of these nine patients developed inflammatory bowel disease (IBD) at 4 and 16 years of age. SLC9A3 encodes Na(+)/H(+) antiporter 3 (NHE3), which is the major intestinal brush-border Na(+)/H(+) exchanger. All mutations were in the NHE3 N-terminal transport domain, and all missense mutations were in the putative membrane-spanning domains. Identified SLC9A3 missense mutations were functionally characterized in plasma membrane NHE null fibroblasts. SLC9A3 missense mutations compromised NHE3 activity by reducing basal surface expression and/or loss of basal transport function of NHE3 molecules, whereas acute regulation was normal. This study identifies recessive mutations in NHE3, a downstream target of GC-C, as a cause of CSD and implies primary basal NHE3 malfunction as a predisposition for IBD in a subset of patients.

The gene cluster of aureocyclicin 4185: the first cyclic bacteriocin of Staphylococcus aureus

Staphylococcus aureus 4185 was previously shown to produce at least two bacteriocins. One of them is encoded by pRJ101. To detect the bacteriocin-encoding gene cluster, an ~9160 kb region of pRJ101 was sequenced. In silico analyses identified 10 genes (aclX, aclB, aclI, aclT, aclC, aclD, aclA, aclF, aclG and aclH) that might be involved in the production of a novel cyclic bacteriocin named aureocyclicin 4185. The organization of these genes was quite similar to that of the gene cluster responsible for carnocyclin A production and immunity. Four putative proteins encoded by these genes (AclT, AclC, AclD and AclA) also exhibited similarity to proteins encoded by cyclic bacteriocin gene clusters. Mutants derived from insertion of Tn917-lac into aclC, aclF, aclH and aclX were affected in bacteriocin production and growth. AclX is a 205 aa putative protein not encoded by the gene clusters of other cyclic bacteriocins. AclX exhibits 50 % similarity to a permease and has five putative membrane-spanning domains. Transcription analyses suggested that aclX is part of the aureocyclicin 4185 gene cluster, encoding a protein required for bacteriocin production. The aclA gene is the structural gene of aureocyclicin 4185, which shows 65 % similarity to garvicin ML. AclA is proposed to be cleaved off, generating a mature peptide with a predicted Mr of 5607 Da (60 aa). By homology modelling, AclA presents four α-helices, like carnocyclin A. AclA could not be found at detectable levels in the culture supernatant of a strain carrying only pRJ101. To our knowledge, this is the first report of a cyclic bacteriocin gene cluster in the genus Staphylococcus.

Isolation and characterization of LHT-type plant amino acid transporter gene from Panax ginseng Meyer

A lysine histidine transporter (LHT) cDNA was isolated and characterized from the roots of Panax ginseng, designated PgLHT. The cDNA is 1,865 bp with an open reading frame that codes for a protein with 449 amino acids and a calculated molecular mass of 50.6 kDa with a predicted isoelectric point of 8.87. Hydropathy analysis shows that PgLHT is an integral membrane protein with 9 putative membrane-spanning domains. Multiple sequence alignments show that PgLHT shares a high homology with other plant LHTs. The expression profile of the gene was investigated by real-time quantitative polymerase chain reaction during various chemical treatments. PgLHT was up-regulated in the presence of abscisic acid, salicylic acid, methyl jasmonate, NaCl, and amino acids. To further explore the function of PgLHT gene, full-length cDNA of PgLHT was introduced into P. ginseng by Agrobacterium rhizogenes A4. The overexpression of PgLHT in the hairy roots led to an obviously increase of biomass compared to the controls, and after addition of the amino acids, the overexpressed-PgLHT hairy roots grew more rapidly than untreated controls during early stage of the culture cycle. The results suggested that the PgLHT isolated from ginseng might have role in the environmental stresses and growth response.

Cloning and Characterization of the Gene Cluster Involved in the Production of the Circular Bacteriocin Carnocyclin A

Carnocyclin A is a circular bacteriocin of 60 amino acids produced by Carnobacterium maltaromaticum UAL307. A region of 12kb that contained the structural gene for carnocyclin A, cclA, was sequenced using a fosmid library, and 10 genes were identified that could be responsible for carnocyclin A production and immunity. Five of those genes, cclBITCD, were found upstream of cclA: one encodes a protein containing a conserved ATP-binding domain and four encode proteins with putative membrane-spanning domains. CclC shows homology with a family of membrane proteins that contain the domain of unknown function 95 (DUF95). Downstream of cclA four additional genes, cclEFGH, were identified that show similarity to the last four genes, as-48EFGH, of the enterocin AS-48 bacteriocin gene cluster. CclFGH shows sequence homology with As-48FGH. Transformation of C. maltaromaticum UAL26 with cclBITCDA resulted in production of carnocyclin A, indicating that these genes form the minimal requirement for the secretion of fully matured bacteriocin. cclI encodes for a small hydrophobic protein with a high pI, which are characteristic features of known immunity proteins for other circular bacteriocins. Indeed, cloning of cclI behind a constitutive promoter in UAL26 resulted in immunity although the level of resistance was lower than that of UAL26 containing cclBITCDA, indicating that CclI alone is not enough to confer full immunity to carnocyclin A.

α-Catulin CTN-1 is required for BK channel subcellular localization in C. elegans body-wall muscle cells

The BK channel, a voltage- and Ca(2+)-gated large-conductance potassium channel with many important functions, is often localized at specific subcellular domains. Although proper subcellular localization is likely a prerequisite for the channel to perform its physiological functions, little is known about the molecular basis of localization. Here, we show that CTN-1, a homologue of mammalian α-catulin, is required for subcellular localization of SLO-1, the Caenorhabditis elegans BK channel α-subunit, in body-wall muscle cells. CTN-1 was identified in a genetic screen for mutants that suppressed a lethargic phenotype caused by expressing a gain-of-function (gf) isoform of SLO-1. In body-wall muscle cells, CTN-1 coclusters with SLO-1 at regions of dense bodies, which are Z-disk analogs of mammalian skeletal muscle. In ctn-1 loss-of-function (lf) mutants, SLO-1 was mislocalized in body-wall muscle but its transcription and protein level were unchanged. Targeted rescue of ctn-1(lf) in muscle was sufficient to reinstate the lethargic phenotype in slo-1(gf);ctn-1(lf). These results suggest that CTN-1 plays an important role in BK channel function by mediating channel subcellular localization.

Identification and Comparative Functional Characterization of a New Human Riboflavin Transporter hRFT3 Expressed in the Brain1–3

We isolated cDNA coding a new human riboflavin transporter (hRFT)3, which exhibits 86.7 and 44.1% amino acid identity with hRFT1 and hRFT2, respectively. It was predicted to have 10 putative membrane-spanning domains. The functional characteristics of hRFT3 were examined and compared with those of its isoforms, hRFT1 and hRFT2. Realtime PCR revealed that hRFT3 mRNA was strongly expressed in the brain and salivary gland. hRFT1 mRNA was strongly expressed in the placenta and small intestine, whereas hRFT2 mRNA was most abundantly expressed in the testis and strongly in the small intestine and prostate. hRFT-mediated uptake of [3H]riboflavin was evaluated using human embryonic kidney 293 cells transiently transfected with the cDNA coding each hRFT. The apparent Michaelis-Menten constants of hRFT1, hRFT2, and hRFT3 for riboflavin were 1.38, 0.98, and 0.33 μmol/L, respectively. The hRFTmediated [3H]riboflavin uptake was independent of extracellular Na+ and Cl−. Specific uptake of [3H]riboflavin by hRFT2, but not hRFT1 and hRFT3, decreased as extracellular pH was changed from 5.4 to 8.4. The substrate specificities of the hRFT family were similar. hRFT-mediated uptake of [3H]riboflavin was inhibited by some riboflavin analogs, but not D-ribose, organic ions, or other vitamins. The newly isolated hRFT3 may play an important role in brain riboflavin homeostasis. Its amino acid sequence and functional characteristics are similar to those of hRFT1, but not hRFT2. J. Nutr. 140: 1220–1226, 2010.

Abstract 526: Tetraspanin CD81/TAPA-1 up-regulates MT1-MMP involved in melanoma cell motility through Akt-dependent Sp1 activation signaling pathways

Abstract Tetraspanin CD81 is a member of transmembrane 4 superfamily proteins that have four putative membrane-spanning domains, short C- and N-terminal cytoplasmic domains, two extracellular loops and small intracellular loop. Tetraspanin CD81 is involved in cell adhesion, cell proliferation, motility and metastasis. In this study, to reveal the role of CD81 in tumor invasion, we examined the effect of CD81 overexpression on invasive phenotype in human melanoma cells. We also investigated the intracellular signaling pathways mediated by CD81 involved in these invasive phenotypes. For this study, we generated MelJuSo human melanoma cell lines stably expressing CD81. Cell invasion and motility of the CD81-transfected cells was enhanced significantly as compared to control transfectant cells lacking CD81 expression. In addition, MT1-MMP expression is up-regulated in CD81 transfectant cells as compared to control transfectant cells. MT1-MMP knockdown in CD81 transfectant cells abolished the increase of cell motility by CD81 expression, suggesting the role of MT1-MMP in CD81-induced cell motility. Through the studies using chemical inhibitors and siRNAs for gene knockdown, we found that Akt/PI3K signaling pathway is involved in CD81-mediated MT1-MMP expression. The enhancement of cell motility by CD81 expression was abolished by knockdown of Akt. Finally, we found the involvement of Sp1 transcription factor in the induction of MT1-MMP expression mediated by CD81. Sp1 binding site in MT1-MMP gene promoter was indispensable for the induction of MT1-MMP expression by CD81. Electrophoretic mobility shift assay confirmed the involvement of Sp1 in MT1-MMP expression by CD81. Taken together, these results suggest that tetraspanin CD81 stimulates melanoma cell invasion and motility by up-regulating MT1-MMP expression through the activation of AKT signaling pathways and Sp1 transcription factor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 526.

Molecular cloning, tissue distribution and ontogenetic expression of sodium proton exchanger isoform 2 (NHE-2) mRNA in the small intestine of pigs

Molecular cloning, tissue distribution and ontogenetic regulation of sodium/proton exchanger isoform 2 (NHE-2) mRNA expression were evaluated in the pig small intestine during postnatal development. The 2872-bp porcine full cDNA sequence of the NHE-2 (EF672046) cloned in this study showed 80% and 70% homology with known human and mouse gene sequence, respectively. Hydrophobic prediction suggests 13 putative membrane-spanning domains within porcine NHE-2. The porcine NHE-2 mRNA was detected in the brain, liver, kidney, heart, lung, small intestine and muscle. The small intestine had the highest NHE-2 mRNA abundance and the brain, lung and liver had the lowest NHE-2 mRNA abundance (P < 0.05). Along the longitudinal axis, the duodenum had the highest NHE-2 mRNA abundance and the ileum and colon had the lowest NHE-2 mRNA abundance (P < 0.05). The NHE-2 mRNA level was increased from day 1 to day 26 in the duodenum (P < 0.05) and dropped dramatically on day 30 (P < 0.05). There is no difference between day 1 and day 7 (P > 0.05). After day 30, the NHE-2 mRNA level remained the same except on day 90 (P > 0.05). The mRNA expression of NHE-2 was not only differentially regulated by age but also differentially distributed along the small intestine of piglets at early stages and growing stages of life, which may contribute to changes in NHE activity.

Molecular Cloning, Tissue Distribution and Ontogenetic Expression of Sodium Proton Exchanger (NHE2) mRNA in the Small Intestine of Pigs

Molecular cloning, tissue distribution and ontogenetic regulation of sodium/proton exchanger isoform 2 (NHE2) mRNA expression were evaluated in the pig small intestine during postnatal development. The 2872‐bp porcine full cDNA sequence of the NHE2 (EF672046) cloned in this study showed 80% and 70% homology with known human and mouse gene sequence, respectively. Hydrophobic prediction suggests 13 putative membrane‐spanning domains within porcine NHE2. The porcine NHE2 mRNA was detected in brain, liver, kidney, heart, lung, small intestine and muscle. The small intestine had the highest NHE2 mRNA abundance and the brain, lung and liver had the lowest NHE2 mRNA abundance (P &lt; 0.05). Along the longitudinal axis, the duodenum had the highest NHE2 mRNA abundance and the ileum and colon had the lowest NHE2 mRNA abundance (P &lt; 0.05). The NHE2 mRNA level was increased from the day 1 to day 26 in the duodenum (P &lt; 0.05) and dropped dramatically on day 30 (P &lt; 0.05). There is no difference between day 1 and day 7 (P &gt; 0.05). After day 30, the NHE2 mRNA level stayed at the same except on day 90 (P &gt; 0.05). The mRNA expression of NHE2 was not only differentially regulated by age but also differentially distributed along the small intestine of piglets at early stages and growing stages of life, which may contribute to changes in NHE activity.

Truncation of cytoplasmic tail of EIAV Env increases the pathogenic necrosis

Equine Infectious Anemia Virus (EIAV), like other lentiviruses, has a transmembrane glycoprotein with an unusually long cytoplasmic tail (CT). Viral envelope (Env) proteins having CT truncations just downstream the putative membrane-spanning domain (PMSD) are assumed to exist among all wild-type budded virions, and also in some cell-adapted strains. To determine whether CT-truncated Env proteins can cause particularly deleterious effects on the Env expressing cells and/or their neighboring cells, plasmids encoding codon-optimized env gene including full-length (pE863) or CT-truncated (pE686* and pE676*) were transiently transfected into 293T cells, respectively. Data from intracellular protein expression and cell death assays revealed that CT-truncated Env, compared to full-length Env, not only induced comparable apoptosis, but also caused much more intensive mitochondria-mediated necrosis that could simultaneously induce significant decrease of intracellular protein expression in the Env expressing cells. Moreover, results from flow cytometric analysis showed that mitochondrial depolarization preceded the caspase activation in cells no matter which env construct was delivered, and indicated that both full-length and CT-truncated Env proteins share a common intrinsic mitochondrial pathway to induce apoptosis. Our results partially elucidate the mechanisms underlying cell death resulting from EIAV pathogenesis.

Identification and expression of XRTN1‐A and XRTN1‐C in Xenopus laevis

Reticulon1, also known as neuroendocrine-specific protein, belongs to the reticulon (RTN) family, whose members possess a conserved reticulon domain and are associated with the endoplasmic reticulum (ER) membrane. Here, we report cloning and expression of Xenopus homologues of Reticulon1-A (XRTN1-A) and -C (XRTN1-C). XRTN1-A and -C contain an open reading frame of 752 and 207 amino acids, respectively, each containing a conserved reticulon domain. Sequence analysis shows that XRTN1 proteins have an ER membrane retention signal and four putative membrane-spanning domains. Reverse transcription-polymerase chain reaction and whole-mount in situ hybridization showed that XRTN1-A is expressed in early neural precursors and differentiating neuronal populations, including the trigeminal placode, olfactory placode, lateral line placode, and otic vesicle. XRTN1-C is expressed in the developing brain and spinal cord. We found that XRTN1-C protein is localized to the ER of Xenopus and mammalian cells and the granules in neurites of primary neurons of the Xenopus spinal cord and rat hippocampus. We also showed that XRTN1-C protein is detected in the heavy membrane fraction, which contains lysosomal and ER-resident proteins, as well as in the nucleus and polysomal fractions of the Xenopus embryo. Finally, we showed that thyroid hormone specifically down-regulates XRTN1-A mRNA in the head of premetamorphic Xenopus tadpoles. Our work characterizes the intracellular roles of XRTN1 during Xenopus neural development.

A Six-membrane-spanning Topology for Yeast and Arabidopsis Tsc13p, the Enoyl Reductases of the Microsomal Fatty Acid Elongating System

The very long chain fatty acids are crucial building blocks of essential lipids, most notably the sphingolipids. These elongated fatty acids are synthesized by a system of enzymes that are organized in a complex within the endoplasmic reticulum membrane. Although several of the components of the elongase complex have recently been identified, little is known about how these proteins are organized within the membrane or about how they interact with one another during fatty acid elongation. In this study the topology of Tsc13p, the enoyl reductase of the elongase system, was investigated. The N and C termini of Tsc13p reside in the cytoplasm, and six putative membrane-spanning domains were identified by insertion of glycosylation and factor Xa cleavage sites at various positions. The N-terminal domain including the first membrane-spanning segment contains sufficient information for targeting to the endoplasmic reticulum membrane. Studies of the Arabidopsis Tsc13p protein revealed a similar topology. Highly conserved domains of the Tsc13p proteins that are likely to be important for enzymatic activity lie on the cytosolic face of the endoplasmic reticulum, possibly partially embedded within the membrane.

A Single Enzyme Catalyzes Both Platelet-activating Factor Production and Membrane Biogenesis of Inflammatory Cells: CLONING AND CHARACTERIZATION OF ACETYL-CoA:LYSO-PAF ACETYLTRANSFERASE

Platelet-activating factor (PAF) is a potent proinflammatory lipid mediator eliciting a variety of cellular functions. Lipid mediators, including PAF are produced from membrane phospholipids by enzymatic cascades. Although a G protein-coupled PAF receptor and degradation enzymes have been cloned and characterized, the PAF biosynthetic enzyme, aceyl-CoA:lyso-PAF acetyltransferase, has not been identified. Here, we cloned lyso-PAF acetyltransferase, which is critical in stimulus-dependent formation of PAF. The enzyme is a 60-kDa microsomal protein with three putative membrane-spanning domains. The enzyme was induced by bacterial endotoxin (lipopolysaccharide), which was suppressed by dexamethasone treatment. Surprisingly, the enzyme catalyzed not only biosynthesis of PAF from lyso-PAF but also incorporation of arachidonoyl-CoA to produce PAF precursor membrane glycerophospholipids (lysophosphatidylcholine acyltransferase activity). Under resting conditions, the enzyme prefers arachidonoyl-CoA and contributes to membrane biogenesis. Upon acute inflammatory stimulation with lipopolysaccharide, the activated enzyme utilizes acetyl-CoA more efficiently and produces PAF. Thus, our findings provide a novel concept that a single enzyme catalyzes membrane biogenesis of inflammatory cells while producing a prophlogistic mediator in response to external stimuli.

Transmembrane topology of ceramide synthase in yeast

Ceramide plays a crucial role as a basic building block of sphingolipids, but also as a signalling molecule mediating cell-fate decisions. Three genes, LAG1, LAC1 and LIP1, have been shown to be required for ceramide synthase activity in Saccharomyces cerevisiae [Guillas, Kirchman, Chuard, Pfefferli, Jiang, Jazwinski and Conzelman (2001) EMBO J. 20, 2655-2665; Schorling, Vallee, Barz, Reizman and Oesterhelt (2001) Mol. Biol. Cell 12, 3417-3427; Vallee and Riezman (2005) EMBO J. 24, 730-741]. In the present study, the topology of the Lag1p and Lac1p subunits was investigated. The N- and C-termini of the proteins are in the cytoplasm and eight putative membrane-spanning domains were identified in Lag1p and Lac1p by insertion of glycosylation and factor Xa cleavage sites at various positions. The conserved Lag motif, potentially containing the active site, is most likely embedded in the membrane. We also present evidence that histidine and aspartic acid residues in the Lag motif are essential for the function of Lag1p in vivo.

Horizontal transfer of the immunoglobulin A1 protease gene (iga) from Streptococcus to Gemella haemolysans

Bacterial IgA1 proteases share the ability to cleave human IgA1 at the hinge region. Nature has developed this trait along at least five independent evolutionary lineages. To obtain further insight into the phylogeny and function of IgA1 proteases, the nucleotide sequence of the iga gene that encodes the IgA1 protease was determined from two Streptococcus mitis strains and one Gemella haemolysans strain. Heterologous expression in Escherichia coli confirmed that the genes encode human IgA1-cleaving activity. IgA1 proteases from Streptococcus and G. haemolysans shared structural features, including a motif typical for zinc-dependent metalloproteases of clan MA(E) family M26 and an N-terminal signal sequence followed by an LPXTG cell-wall-anchor motif and two putative membrane-spanning domains. In addition, they all harboured a repeat region preceding the active site of the protease. In the streptococcal IgA1 proteases, a G5 domain, which has been suggested to bind N-acetylglucosamine, was identified. Conservation of these structures in otherwise diverse proteases suggests that they are essential to the biological function of the enzyme. The phylogenetic distribution of homologous iga genes and conservation of gene order in the iga gene region in different Streptococcus species, combined with the sequence homologies, strongly suggest that the iga gene is more ancient in Streptococcus than in G. haemolysans, and therefore that the IgA1 protease gene was transferred from Streptococcus to G. haemolysans.

Structure and role of the first membrane‐spanning domain of the epithelial sodium channel

Epithelial Na+ Channels (ENaC) are comprised of three subunits that have been proposed to be arranged in either an α2βγ or a higher ordered configuration. Each subunit has two putative membrane-spanning domains (M1 and M2), intracellular N- and C- termini, and a large extracellular loop. We have systematically mutated each M1 residue in the α subunit to Trp, and endogenous M1 Trp’s to Ala in order to experimentally determine the secondary structure of M1, and the role of M1 in ENaC pore formation. Na+ current and Li+:Na+ selectivity of M1 mutants varied with a periodicity consistent with an α-helical secondary structure. Throughout M1, changes in channel activity correlated inversely with changes in Li+:Na+ selectivity, suggesting changes in ion permeability. Preliminary surface expression data suggests that the changes induced by these mutations are caused by changes in the single channel properties of ENaC. Our results provide the first experimental evidence for the helical structure of M1. They also suggest that M1 may slightly unwind or have a kink at a Gly in the middle of M1. A model in which M1 from each subunit forms a packing shell around the pore-forming M2 helices may account for our observations. This work was supported in part by grants from the NIH (DK054354). OBK was supported by NIH grants DK061296 and DK066883. KMB was supported by NIH grant GM60582.

Scallop DMT functions as a Ca 2+ transporter

We identified a DMT (divalent metal transporter) homologous protein that functions as a Ca 2+ transporter. Scallop DMT cDNA encodes a 539-amino-acid protein with 12 putative membrane-spanning domains and has a consensus transport motif in the fourth extracellular loop. Since its mRNA is significantly expressed in the gill and intestine, it is assumed that scallop DMT transports Ca 2+ from seawater by the gill and from food by the intestine. Scallop DMT lacks the iron-responsive element commonly found in iron-regulatory proteins, suggesting that it is free of the post-transcriptional regulation from intracellular Fe 2+ concentration. Scallop DMT distinctly functions as a Ca 2+ transporter unlike other DMTs, however, it also transports Fe 2+ and Cd 2+ similar to them.