Sequence, structure, and chromosomal mapping of the mouse Lgals6 gene, encoding galectin-6

In the accompanying paper (Gitt, M. A., Colnot, C., Poirier, F., and Barondes, S. H., and Leffler, H. (1998) J. Biol. Chem. 273, 2954-2960), we reported that mouse gastrointestinal tract specifically expresses two closely related galectins, galectins-4 and -6, each with two carbohydrate recognition domains in the same peptide. Here, we report the isolation, characterization, and chromosomal mapping of the complete mouse Lgals6 gene, which encodes galectin-6, and of a fragment of a distinct gene, Lgals4, which encodes galectin-4. The coding sequence of galectin-6 is specified by eight exons. The upstream region contains two putative promoters. Both Lgals6 and the closely related Lgals4 are clustered together about 3.2 centimorgans proximal to the apoE gene on mouse chromosome 7. The syntenic human region is 19q13.1-13.3.     

Is the continuity of the domains required for the correct folding of a two-domain protein?

The role of domains in protein folding has been widely studied and discussed. Nevertheless, it is not clear whether the continuity of the domains in a protein is an essential requirement in determining the folding pathway. Previous studies have shown that the isolated structural domains of the two-domain monomeric enzyme, yeast phosphoglycerate kinase (yPGK), are able to fold independently into a quasinative structure, but they neither reassociate nor generate a functional enzyme [Minard, P., Hall, L., Betton, J. M., Missiakas, D., & Yon, J. M. (1989) Protein Eng. 3, 55-60; Fairbrother, W. J., Bowen, D., Hall, L., Williams, R. J. P. (1989) Eur. J. Biochem. 184, 617-625; Missiakas, D., Betton, J. M., Minard, P., & Yon, J. M. (1990) Biochemistry 29, 8683-8689]. In the present work, two circularly permuted variants of the yPGK gene were constructed. The natural adjacent chain termini were directly connected and the new extremities were created within the N-domain (at residues 71 and 72) or the C-domain (at residues 291 and 292), respectively. These two proteins were overexpressed and purified. They exhibit 14% and 23% of the wild-type enzyme activity, respectively. The two mutants fold in a compact conformation with slight changes in the secondary and tertiary structure probably related to the presence of a heterogeneous population of molecules. The unfolding studies reveal a large decrease in stability. From the present data it appears that, although the circular permutations induce some perturbations in the structure and stability of the protein, the continuity of the domains is not required for the protein to reach a native-like and functional structure.     

Tmp21 and p24A, two type I proteins enriched in pancreatic microsomal membranes, are members of a protein family involved in vesicular trafficking

We report here on the isolation, cloning, and expression of two Mr 21,000 proteins from rat pancreatic acinar cells, the rat-Tmp21 (transmembrane protein, Mr 21,000) and the rat-p24A. Both proteins are transmembrane proteins with type I topology and share weak but significant homology to one another (23% identity). We further show the cloning and characterization of the human homologs, hum-Tmp21, which is expressed in two variants (Tmp21-I and Tmp21-II), and hum-p24A. Tmp21 proteins and p24A have highly conserved COOH-terminal tails, which contain motifs related to the endoplasmic reticulum retention and retrieval consensus sequence KKXX. The rat-p24 sequence is identical to the hamster CHOp24, a recently characterized component of coatomer-coated transport vesicles, which defines a family of proteins (called the p24 family) proposed to be involved in vesicular transport processes (Stamnes, M. A., Craighead, M. W., Hoe, M. H., Lampen, N., Geromanos, S., Tempst, P., and Rothman, J. E.(1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8011-8015). Sequence alignment and structural features identify the Tmp21 protein as a new member of this p24 family. Northern analysis of various tissues indicates that the Tmp21 proteins and the p24A protein are ubiquitously expressed. The integral membrane components Tmp21 and p24A are localized in microsomal membranes, zymogen granule membranes, and the plasma membrane and are absent from the cytosol. Both p24A and Tmp21 show weak homology to the yeast protein Emp24p, which recently has been shown to be involved in secretory protein transport from the endoplasmic reticulum to the Golgi apparatus. This leads us to conclude that the receptor-like Tmp21 and p24A are involved in vesicular targeting and protein transport.     

The carboxyl terminus of B class ephrins constitutes a PDZ domain binding motif

Ephrin B proteins function as ligands for B class Eph receptor tyrosine kinases and are postulated to possess an intrinsic signaling function. The sequence at the carboxyl terminus of B-type ephrins contains a putative PDZ binding site, providing a possible mechanism through which transmembrane ephrins might interact with cytoplasmic proteins. To test this notion, a day 10.5 mouse embryonic expression library was screened with a biotinylated peptide corresponding to the carboxyl terminus of ephrin B3. Three of the positive cDNAs encoded polypeptides with multiple PDZ domains, representing fragments of the molecule GRIP, the protein syntenin, and PHIP, a novel PDZ domain-containing protein related to Caenorhabditis elegans PAR-3. In addition, the binding specificities of PDZ domains previously predicted by an oriented library approach (Songyang, Z., Fanning, A. S., Fu, C., Xu, J., Marfatia, S. M., Chishti, A. H., Crompton, A., Chan, A. C., Anderson, J. M., and Cantley, L. C. (1997) Science 275, 73-77) identified the tyrosine phosphatase FAP-1 as a potential binding partner for B ephrins. In vitro studies demonstrated that the fifth PDZ domain of FAP-1 and full-length syntenin bound ephrin B1 via the carboxyl-terminal motif. Lastly, syntenin and ephrin B1 could be co-immunoprecipitated from transfected COS-1 cells, suggesting that PDZ domain binding of B ephrins can occur in cells. These results indicate that the carboxyl-terminal motif of B ephrins provides a binding site for specific PDZ domain-containing proteins, which might localize the transmembrane ligands for interactions with Eph receptors or participate in signaling within ephrin B-expressing cells.     

Cloning, expression and reconstitution of the trypanothione-dependent peroxidase system of Crithidia fasciculata

Tyrosine phosphoproteins of size 115-120 kDa were purified from membranes of chicken embryo fibroblasts (CEF) infected with Rous sarcoma virus (RSV). A mouse was immunized with these proteins, and the immune serum was used to screen a CEF cDNA expression library. A highly immunoreactive clone (KS5) was identified and characterized. The cDNA of this clone is 2.3 kb in length with a short 5' UTR and a single major open reading frame (ORF) encoding a polypeptide of 719 amino acids, with a calculated molecular weight of 81.1 kDa. The encoded protein contains an amino terminal PDZ domain, followed by a predicted coiled-coil region, a PEST domain, and a carboxy-terminal SAM domain. Consensus sequence motifs for tyrosine phosphorylation are also present, as are consensus sequences for the binding of SH2 and PDZ domains. Antisera from mice immunized with bacterially expressed fragments of the KS5 protein recognized proteins of size 230, 116, and 65 kDa in CEF. In other chicken embryo tissues, a 116-kDa species was the predominant protein recognized. The 116-kDa species is tyrosine-phosphorylated in RSV-CEF. The presence of PDZ and SAM domains in the KS5 protein suggests that it may act as a molecular adaptor, promoting and relaying information in a signal transduction pathway. It is a member of a family of related proteins, all of which have a highly conserved PDZ domain adjacent to a coiled-coil region. Two other members of this family are the neuronal proteins spinophilin (Allen, P.B., Ouimet, C.C., Greengard, P., 1997. Spinophilin, a novel protein phosphatase 1 binding protein localized to dendritic spines. Proc. Natl. Acad. Sci. USA 94, 9956-9961) and neurabin (Nakanishi, H., Obaishi, H., Satoh, A., Wada, M., Mandai, K., Satoh, K., Nishioka, H., Matsuura, Y., Mizoguchi, A. , Takai, Y., 1997. Neurabin: A novel neural tissue-specific actin filament-binding protein involved in neurite formation. J. Cell Biol. 139, 951-961).     

TrwD, a protein encoded by the IncW plasmid R388, displays an ATP hydrolase activity essential for bacterial conjugation

A 1.7-kilobase pair segment from the conjugative transfer region of plasmid R388 DNA was cloned and sequenced. It contained trwD, a gene essential for plasmid R388 conjugation, for expression of the conjugative W-pilus and for sensitivity to phage PRD1. The deduced amino acid sequence of TrwD showed homology to the PulE/VirB11 superfamily of potential ATPases involved in various types of transport processes. A fusion of trwD with the glutathione S-transferase (GST) was constructed, and the resulting fusion protein was purified from overproducing bacteria. Factor Xa hydrolysis of GST-TrwD and further purification rendered TrwD protein with more than 95% purity. Antibodies raised against TrwD localized it both in the soluble fraction and in the outer membrane of Escherichia coli. TrwD is probably a peripheral outer membrane protein because it could be solubilized by increasing salt concentration to 0.5 M NaCl in the lysis buffer. Both purified GST-TrwD and TrwD could hydrolize ATP. ATPase activity increased 2-fold in the presence of detergent-phospholipid mixed micelles. To study the importance of the nucleotide-binding site, Walker box A (GXXGXGK(T/S)), present in TrwD, the conserved lysine residue was replaced by glutamine. The mutant protein, expressed and purified under the same conditions as the wild type, did not exhibit ATPase activity. TrwD(K203Q) was not able to complement the mutation in trwD of the R388 mutant plasmid, suggesting the essentiality of the ATPase activity of the protein in the conjugative process. Furthermore, the dominant character of this mutation suggested that GST-TrwD(K432Q) was still able to interact either with itself or with other component(s) of the conjugative machinery.     

Distribution of genes encoding resistance to streptogramin A and related compounds among staphylococci resistant to these antibiotics

The levels of resistance to pristinamycin (Pt) and to its major constituents, pristinamycin IIA and IB (PIIA and PIB, respectively; classified as streptogramins A and B, respectively) were determined for 126 staphylococcal isolates. The results suggest tentative susceptibility breakpoints of < or = 2, < or = 8, and < or = 0.5 microgram/ml for PIIA, PIB, and Pt, respectively. Fifty-six isolates that were inhibited by > or = 4 micrograms of PIIA per ml were investigated for the presence of staphylococcal genes encoding resistance to PIIA (vga, vat, and vatB) and PIB (vgb). None of these genes was found in the 4 isolates inhibited by 4 micrograms of PIIA per ml or in 4 of the other 52 isolates tested. The remaining 48 isolates harbored plasmids carrying vatB and vga or combinations of genes (vga-vat-vgb or vga-vat). The absence of any known PIIA resistance gene from the four Staphylococcus aureus isolates inhibited by > or = 8 micrograms of PIIA per ml suggests that there is at least one PIIA resistance mechanism in staphylococci that has not yet been characterized.     

Synapsins I and II are ATP-binding proteins with differential Ca2+ regulation

Synapsins I and II are abundant phosphoproteins that are localized to synaptic vesicles and have essential functions in regulating synaptic vesicle exocytosis. Synapsins contain a single evolutionarily conserved, large central domain, the C-domain, that accounts for the majority of their sequences. Unexpectedly, the crystal structure of the C-domain from synapsin I revealed that it is structurally closely related to several ATPases despite the absence of sequence similarities (Esser, L., Wang, C.-R., Hosaka, M., Smagula, C. S., Südhof, T. C., and Deisenhofer, J. (1998) EMBO J., in press). We now show that the C-domains of both synapsin I and synapsin II constitute high affinity ATP-binding modules. The two C-domains exhibit similar ATP affinities but are differentially regulated; ATP binding to synapsin I is Ca(2+)-dependent whereas ATP binding to synapsin II is Ca(2+)-independent. In synapsin I, the Ca2+ requirement for ATP binding is mediated by a single, evolutionarily conserved glutamate residue (Glu373) at a position where synapsin II contains a lysine residue. Exchange of Glu373 for lysine converts synapsin I from a Ca(2+)-dependent protein into a Ca(2+)-independent ATP-binding protein. Our studies suggest that synapsins I and II function on synaptic vesicles as ATP-binding proteins that are differentially regulated by Ca2+.     

pMH2, a small plasmid bearing the nif gene cluster of Enterobacter agglomerans 333 as an excisable cassette

A small plasmid containing the entire nif gene cluster of Enterobacter agglomerans 333 as an excisable cassette has been constructed, using pACYC177 as a vector. Two cosmid clones taken from a gene library of E. agglomerans plasmid pEA3 were used as a source of nif genes. A SmaI fragment of peaMS2-2, containing the H,D,K,Y,E,N,X,U,S,V,W,Z,M,L,A and B genes and an ApaI fragment of peaMS2-16 containing nif A,B,Q,F and J were selected to construct pMH2. The resulting plasmid of 33 kb carries the complete nif gene cluster as a nif cassette on a single XbaI fragment. The nif construct pMH2 in Escherichia coli strains has significant nitrogenase activity compared to wild-type E. agglomerans 333. The nif gene cluster construct was found to be very stable.     

Nineteen baculovirus open reading frames, including LEF-12, support late gene expression

A set of 18 plasmid subclones of the Autographa californica nuclear polyhedrosis virus genome, each containing an identified late expression factor gene (lef), supports expression from a late viral promoter in transient expression assays in the SF-21 cell line derived from Spodoptera frugiperda. We have constructed a further set of plasmids in which each lef open reading frame (ORF) is controlled by the Drosophila melanogaster heat shock protein 70 (hsp70) promoter and epitope tagged. Failure of this set of plasmids to support transient late gene expression, and the inability of the p47 ORF to replace the p47-containing plasmid supplied in the lef plasmid library, led to the identification of a 19th late expression factor gene (lef-12) located adjacent to the p47 gene. The sequence of lef-12 is predicted to encode a protein of 21 kDa with no homology to any previously identified protein. The set of 19 hsp70-controlled lef ORFs (HSEpiHis lef library) supports transient expression from a late viral promoter. lef-12 did not affect expression from an early baculovirus promoter. In TN-368 cells, which are also permissive for virus replication, lef-12 provided a stimulatory effect but did not appear to be essential.     

Reduction of CDP-diacylglycerol synthase activity results in the excretion of inositol by Saccharomyces cerevisiae

A yeast mutant, cdg1, was isolated on the basis of an inositol excretion phenotype. This mutant exhibited pleiotropic deficiencies in phospholipid biosynthesis, including reduced levels of CDP-diacylglycerol (DAG) synthase activity (Klig, L. S., Homann, M. J., Kohlwein, S. D., Kelley, M. J., Henry, S. A., and Carman, G. M. (1988) J. Bacteriol. 170, 1878-1886). In this study we present evidence that the molecular basis for the inositol excretion phenotype is a G305/A305 point mutation (Cys102 --> Tyr substitution) within the CDS1 gene (encodes CDP-DAG synthase) of this mutant. Expression of CDP-DAG synthase activity from a plasmid-borne copy of the CDS1 gene in the cdg1 mutant was not down-regulated, and this expression also corrected the inositol excretion phenotype. Introduction of the above mutated gene (CDS1*) controlled by its endogenous promoter on a single copy plasmid into a cds1-null background reconstituted a transformant with the cdg1 phenotype, including reduced CDP-DAG synthase activity, elevated phosphatidylserine synthase activity, and inositol excretion into the growth medium. Expression of CDS1* in a single copy in the cdg1 mutant raised CDP-DAG synthase activity from 15 to 30% of derepressed wild-type yeast levels but still did not correct the inositol excretion phenotype. CDP-DAG synthase activity was not regulated in response to precursors of phospholipid biosynthesis in the cdg1 mutant either with or without a trans copy of the CDS1* gene. An open reading frame was identified 5' to the CDS1 locus, YBR0314, which also resulted in inositol excretion when present in trans in multiple copies.     

A novel DNA binding and nuclease activity in domain III of Mu transposase: evidence for a catalytic region involved in donor cleavage

The Mu A protein is a 75 kDa transposase organized into three structural domains. By severing the C-terminal region (domain III) from the remainder of the protein, we unmasked a novel non-specific DNA binding and nuclease activity in this region. Deletion analysis localized both activities to a 26 amino acid stretch (aa 575-600) which remarkably remained active in DNA binding and cleavage. The two activities were shown to be tightly linked by site-directed mutagenesis. To study the importance of these activities in the transposition process, an intact mutant transposase lacking the DNA binding and nuclease activity of domain III was constructed and purified. The mutant transposase was indistinguishable from wild-type Mu A in binding affinity for both the Mu ends and the enhancer, and in strand transfer activity when the cleavage step was bypassed. In contrast, the mutant transposase displayed defects in both synapsis and donor cleavage. Our results strongly suggest that the 26 amino acid region in domain III carries catalytic residues required for donor DNA cleavage by Mu A protein. Furthermore, our data suggest that an active site for donor cleavage activity in the Mu tetramer is assembled from domain II (metal ion binding) in one A monomer and domain III (DNA cleavage) in a separate A monomer. This proposal for active site assembly is in agreement with the recently proposed domain sharing model by Yang et al. (Yang, J.Y., Kim, K., Jayaram, M. and Harshey, R.M. [1995] EMBO J., 14, 2374-2384).     

Phosphorylation of the periplasmic binding protein in two transport systems for arginine incorporation in Escherichia coli K-12 is unrelated to the function of the transport system

In Escherichia coli K-12, the accumulation of arginine is mediated by two distinct periplasmic binding protein-dependent transport systems, one common to arginine and ornithine (AO system) and one for lysine, arginine, and ornithine (LAO system). Each of these systems includes a specific periplasmic binding protein, the AO-binding protein for the AO system and the LAO-binding protein for the LAO system. The two systems include a common inner membrane transport protein which is able to hydrolyze ATP and also phosphorylate the two periplasmic binding proteins. Previously, a mutant resistant to the toxic effects of canavanine, with low levels of transport activities and reduced levels of phosphorylation of the two periplasmic binding proteins, was isolated and characterized (R. T. F. Celis, J. Biol. Chem. 265:1787-1793, 1990). The gene encoding the transport ATPase enzyme (argK) has been cloned and sequenced. The gene possesses an open reading frame with the capacity to encode 268 amino acids (mass of 29.370 Da). The amino acid sequence of the protein includes two short sequence motifs which constitute a well-defined nucleotide-binding fold (Walker sequences A and B) present in the ATP-binding subunits of many transporters. We report here the isolation of canavanine-sensitive derivatives of the previously characterized mutant. We describe the properties of these suppressor mutations in which the transport of arginine, ornithine, and lysine has been restored. In these mutants, the phosphorylation of the AO- and LAO-binding proteins remains at a low level. This information indicates that whereas hydrolysis of ATP by the transport ATPase is an obligatory requirement for the accumulation of these amino acids in E. coli K-12, the phosphorylation of the periplasmic binding protein is not related to the function of the transport system.