Extra Cysteine Residue Research Articles

Analysis of expressed sequence tags from a single wheat cultivar facilitates interpretation of tandem mass spectrometry data and discrimination of gamma gliadin proteins that may play different functional roles in flour

BackgroundThe gamma gliadins are a complex group of proteins that together with other gluten proteins determine the functional properties of wheat flour. The proteins have unusually high levels of glutamine and proline and contain large regions of repetitive sequences. While most gamma gliadins are monomeric proteins containing eight conserved cysteine residues, some contain an additional cysteine residue that enables them to be linked with other gluten proteins into large polymers that are critical for flour quality. The ability to differentiate among the gamma gliadins is important for studies of wheat flour quality because proteins with similar sequences can have different effects on functional properties.ResultsThe complement of gamma gliadin genes expressed in the wheat cultivar Butte 86 was evaluated by analyzing publicly available expressed sequence tag (EST) data. Eleven contigs were assembled from 153 Butte 86 ESTs. Nine of the contigs encoded full-length proteins and four of the proteins contained nine cysteine residues. Only one of the encoded proteins was a perfect match with a sequence reported in NCBI. Contigs from four different publicly available EST assemblies encoded proteins that were perfect matches with some, but not all, of the Butte 86 gamma gliadins and the complement of identical proteins was different for each assembly. A specialized database that included the sequences of Butte 86 gamma gliadins was constructed for identification of flour proteins by tandem mass spectrometry (MS/MS). In a pilot experiment, proteins corresponding to six Butte 86 gamma gliadin contigs were distinguished by MS/MS, including one containing the extra cysteine residue. Two other proteins were identified as one of two closely related Butte 86 proteins but could not be distinguished unequivocally. Unique peptide tags specific for Butte 86 gamma gliadins are reported.ConclusionsInclusion of cultivar-specific gamma gliadin sequences in databases maximizes the number and quality of peptide identifications and increases sequence coverage of these gamma gliadins by MS/MS. This approach makes it possible to distinguish closely related proteins, to associate individual proteins with sequences of specific genes, and to evaluate proteomic data in a biological context to better address questions about wheat flour quality.

Identification of canine natural CD3-positive T cells expressing an invariant T-cell receptor alpha chain

A representative T-cell subset exclusively using an invariant TCRα chain (iTCRα) is natural killer T (NKT) cells which are becoming an emerging topic for cancer and immune disorder in humans and mice. However, NKT cells in dogs have not yet been identified. In this study, CD3 + T-lymphocyte population reactive to α-galactosylceramide-loaded mouse CD1d (α-GalCer/CD1d) were identified with flow cytometric analysis in mononuclear cells from spleen, liver, and peripheral blood of a dog with percentages of 0.028%, 0.045%, and 0.004%, respectively. Using cDNA library synthesized from mRNAs of the α-GalCer/CD1d reactive CD3 + lymphocytes in the spleen cells, molecular analysis of canine iTCRα was carried out. Consequently, Variable (Vα) and Joining (Jα) regions of iTCRα cDNA were found to be homogeneous to both mouse Vα14–Jα281 and human Vα24–JαQ. Characteristic features of iTCRα of NKT cells, such as the amino acid sequence of complementarity-determining region (CDR) 3 and extra cysteine residue, were well conserved among dogs, mice, and humans. In quantitative real-time PCR analysis, relative expression of the canine iTCRα mRNA in α-GalCer/CD1d reactive CD3 + lymphocytes was 271-fold higher than that in CD3 + lymphocytes unbound to α-GalCer/CD1d, indicating that the iTCRα mRNA was preferentially expressed in α-GalCer/CD1d-reactive CD3 + lymphocytes in the dog. Together, the results strongly suggested that α-GalCer/CD1d-binding CD3 + T lymphocytes identified in this study could be considered to be canine NKT cells.

Variant c-type cytochromes as probes of the substrate specificity of the E. coli cytochrome c maturation (Ccm) apparatus

c-type cytochromes are normally characterized by covalent attachment of the iron cofactor haem to protein through two thioether bonds between the vinyl groups of the haem and the thiol groups of a CXXCH (Cys-Xaa-Xaa-Cys-His) motif. In cells, the haem attachment is an enzyme-catalysed post-translational modification. We have previously shown that co-expression of a variant of Escherichia coli cytochrome b(562) containing a CXXCH haem-binding motif with the E. coli Ccm (cytochrome c maturation) proteins resulted in homogeneous maturation of a correctly formed c-type cytochrome. In contrast, in the absence of the Ccm apparatus, the product holocytochrome was heterogeneous, the main species having haem inverted and attached through only one thioether bond. In the present study we use further variants of cytochrome b(562) to investigate the substrate specificity of the E. coli Ccm apparatus. The system can mature c-type cytochromes with CCXXCH, CCXCH, CXCCH and CXXCHC motifs, even though these are not found naturally and the extra cysteine residue might, in principle, disrupt the biogenesis proteins which must interact intricately with disulfide-bond oxidizing and reducing proteins in the E. coli periplasm. The Ccm proteins can also attach haem to motifs of the type CX(n)CH where n ranges from 2 to 6. For n=3 and 4, the haem attachment was correct and homogeneous, but for higher values of n the holocytochromes displayed oxidative addition of sulfur and/or oxygen atoms associated with the covalent haem-attachment process. The implications of our observations for the haem-attachment reaction, for genome analyses and for the substrate specificity of the Ccm system, are discussed.

Molecular characterization and comparative analysis of four new genes from Sec2 locus encoding 75K γ-secalins of rye species

Using a PCR-based strategy, four new 75K γ-secalin genes were isolated from Secale cereale, Secale vavilovii, Secale sylvestre, and Secale strictum in genus Secale (rye). Based on amino acid sequences, the primary structure of the 75K γ-secalin subunits was demonstrated, which was composed of four main structural regions: (a) a conservative 19 amino acids signal peptide, (b) a steady short N-terminal region of 12 amino acids containing a cysteine residue, (c) a repetitive domain, which began with the conservative tetrapeptides PQ 3 and was rich in glutamine and proline. PFPQ 1−2(PQQ) 1−2 was the core repeat motif in the repetitive region. Besides amino acid substitutions, this region showed variations in length due to the insertion and deletion events. In the repetitive region of EF432549 ( Secale strictum), there were two octapeptides (PFPQQPQQ and PVPQQSQQ) insertions. On the contrary, deletion events of two residues (QT) took place in EF432546 ( Secale sylvestre). Accounting for the amino acid replacement, an extra cysteine residue appeared in the repetitive region of EF432546, which did not exist in other genes, and (d) a conserved 143 amino acids C-terminal domain including eight cysteine residues. The implications of the results for quality improvement are discussed.

Mammalian Sec16/p250 Plays a Role in Membrane Traffic from the Endoplasmic Reticulum

Coat protein complex II (COPII)-coated vesicles/carriers, which mediate export of proteins from the endoplasmic reticulum (ER), are formed at special ER subdomains in mammals, termed ER exit sites or transitional ER. The COPII coat consists of a small GTPase, Sar1, and two protein complexes, Sec23-Sec24 and Sec13-Sec31. Sec23-Sec24 and Sec13-Sec31 appear to constitute the inner and the outermost layers of the COPII coat, respectively. We previously isolated two mammalian proteins (p125 and p250) that bind to Sec23. p125 was found to be a mammalian-specific, phospholipase A(1)-like protein that participates in the organization of ER exit sites. Here we show that p250 is encoded by the KIAA0310 clone and has sequence similarity to yeast Sec16 protein. Although KIAA0310p was found to be localized at ER exit sites, subcellular fractionation revealed its predominant presence in the cytosol. Cytosolic KIAA0310p was recruited to ER membranes in a manner dependent on Sar1. Depletion of KIAA0310p mildly caused disorganization of ER exit sites and delayed protein transport from the ER, suggesting its implication in membrane traffic out of the ER. Overexpression of KIAA0310p affected ER exit sites in a manner different from that of p125. Binding experiments suggested that KIAA0310p interacts with both the inner and the outermost layer coat complexes, whereas p125 binds principally to the inner layer complex. Our results suggest that KIAA0310p, a mammalian homologue of yeast Sec16, builds up ER exit sites in cooperation with p125 and plays a role in membrane traffic from the ER.

Self-assembled films of hydrophobin protein HFBIII from Trichoderma reesei

Hydrophobins are a group of small amphiphilic proteins which are known to self-assemble on interfaces. They contain eight conserved cysteine residues, which make four disulfide bridges. A new hydrophobin protein, HFBIII, from the fungus Trichoderma reesei contains one extra cysteine residue, giving the protein a naturally reactive site. The self-assembly of hydrophobin protein HFBIII was studied using grazing-incidence X-ray diffraction and reflectivity. HFBIII self-assembles into a hexagonally ordered monolayer at an air/water interface and also forms crystalline coatings on a silicon substrate. The lattice constants for the hexagonal coatings are a = b = 56.5 A, γ = 120°. The self-assembled structure in the HFBIII film is very similar to those formed by two other T. reesei hydrophobins, HFBI and HFBII.

Characterization of the HMW glutenin subunits from Aegilops searsii and identification of a novel variant HMW glutenin subunit

High molecular weight (HMW) glutenin subunits are conserved seed storage proteins in wheat and related species. Here we describe a more detailed characterization of the HMW glutenin subunits from Aegilops searsii, which is diploid and contains the S(s) genome related to the S genome of Aegilops speltoides and the A, B and D genomes of hexaploid wheat. SDS-PAGE experiments revealed two subunits (one x and one y) for each of the nine Ae. searsii accessions analyzed, indicating that the HMW glutenin subunit gene locus of Ae. searsii is similar to the Glu-1 locus found in wheat in containing both x and y genes. The primary structure of the four molecularly cloned subunits (from two Ae. searsii accessions) was highly similar to that of the previously reported x and y subunits. However, in one accession (IG49077), the last 159 residues of the x subunit (1S(s)x49077), which contained the sequence element GHCPTSPQQ, were identical to those of the y subunit (1S(s)y49077) from the same accession. Consequently, 1S(s)x49077 contains an extra cysteine residue located at the C-terminal part of its repetitive domain, which is novel compared to the x-type subunits reported so far. Based on this and previous studies, the structure and expression of the Glu-1 locus in Ae. searsii is discussed. A hypothesis on the genetic mechanism generating the coding sequence for the novel 1S(s)x49077 subunit is presented.

Cloning and molecular characterization of three novel LMW-i glutenin subunit genes from cultivated einkorn (Triticum monococcum L.)

Three novel low molecular weight (LMW) glutenin subunits from cultivated einkorn (Triticum monococcum L., A(m)A(m), 2n = 2x = 14) were characterized by SDS-PAGE and molecular weights determined by MALDI-TOF-MS. Their coding genes were amplified and cloned with designed AS-PCR primers, revealing three complete gene sequences. All comprised upstream, open reading frame (ORF), downstream and no introns were present. The deduced amino acid sequences showed that all three genes, named as LMW-M1, LMW-M3 and LMW-M5, respectively, belonged to the LMW-i type subunits with the predicted molecular weight between 38.5206 and 38.7028 kDa. They showed high similarity with other LMW-i type genes from hexaploid bread wheats, but also displayed unique features. Particularly, LMW-M5 subunit contained an extra cysteine residue in the C-terminus except for eight conserved cysteines, which resulted from a single-nucleotide polymorphism (SNP) of the T-C transition, namely arginine --> cysteine substitution at position 242 from the N-terminal end. This is the first report that the LMW-i subunit contained nine cysteines residues that could result in a more highly cross-linked and more elastic glutenin suggesting that LMW-M5 gene may associates with good quality properties. In addition, a total of 25 SNPs and one insertions/deletions (InDels) were detected among three LMW-i genes, which could result in significant functional changes in polymer formation of gluten. It is anticipated that these SNPs could be used as reliable genetic markers during wheat quality improvement. The phylogenetic analysis indicated that LMW-i type genes apparently differed from LMW-m and LMW-s type genes and diverged early from the primitive LMW-GS gene family, at about 12.92 million years ago (MYA) while the differentiation of A(m) and A genomes was estimated at 3.98 MYA.

A proteomic approach to verify in vivo expression of a novel γ‐gliadin containing an extra cysteine residue

Gliadins and glutenins are the main protein fractions present in wheat gluten. They are responsible for technological and nutritional quality of wheat based products. In particular, glutenins are mainly responsible for dough visco-elastic properties, whereas gliadins confer extensibility to dough and are the most important factor triggering celiac disease, the major human intolerance to gluten. Gliadins are monomeric proteins, whereas glutenins are polymers stabilized by disulfide bonds. Although they have distinctive structural characteristics, it is possible that some gliadins become part of the glutenin fraction because of mutations that affect cysteine number and distribution. Here, we provide evidence that a naturally mutated gamma-gliadin with an extra cysteine residue is incorporated into the polymeric fraction. This goal was achieved using an integrated approach involving heterologous expression, 2-DE, RP-HPLC and MS.

A high-molecular-weight glutenin subunit gene1Dx2.1from Xinjiang rice wheat

Xinjiang rice wheat (Triticum petropavlovskyi Udacz. et Migush, 2n=6x=42, AABBDD) is one of the endemic Chinese wheats, only distributing in Xinjiang and Xizang (Tibet), China. A novel high-molecular-weight (HMW) glutenin subunit gene 1DX2.1 was isolated and characterized from Xinjiang rice wheat accession Daomai2. The complete open reading frame (ORF) of 1Dx2.1 is 2508 bp, encoding 836 amino acids. The primary structure of 1DX2.1 consists of three distinct domains, a non-repetitive N-terminal domain with 89 residues, a non-repetitive C-terminal domain with 42 residues and a large central repetitive domain with 684 residues. In the N-terminal of 1DX2.1, there is an R (arginine) at position 75, whereas there is a Q (glutamine) in other known x-type subunits. Four cysteine residues are observed in 1DX2.1 with three in the N-terminal region and one in the C-terminal region. The number and distribution of cysteines in 1DX2.1 are identical to those in x-type subunits except for 1DX5, which possesses an extra cysteine residue. Differences between the repetitive domain of 1DX2.1 and those of known HMW subunits resulted from substitutions, insertions or/and deletions involving single or more amino acid residues. The phylogenetic tree, which was constructed on the basis of amino acid sequences, and indicated that 1DX2.1 was highly related to 1Dx2.lt, then to 1DX2 and 1DX5.

The CD8α from sea bass ( Dicentrarchus labrax L.): Cloning, expression and 3D modelling

In this paper we describe the cloning, expression and structural study by modelling techniques of the CD8α from sea bass ( Dicentrarchus labrax L.). The sea bass CD8α cDNA is comprised of 1490 bp and is translated in one reading frame to give a protein of 217 amino acids, with a predicted 26 amino acids signal peptide, a 88 bp 5′-UTR and a 748 bp 3′-UTR. A multiple alignment of CD8α from sea bass with other known CD8α sequences shows the conservation of most amino acid residues involved in the peculiar structural domains found within CD8α's. Cysteine residues that are involved in disulfide bonding to form the V domain are conserved. In contrast, an extra cysteine residue found in most mammals in this region is not present in sea bass. The transmembrane and cytoplasmic regions are the most conserved regions within the molecule in the alignment analysis. However, the motif (CXCP) that is thought to be responsible for binding p56 lck is missing in the sea bass sequence. Phylogenetic analysis conducted using amino acid sequences showed that sea bass CD8α grouped with other known teleost sequences and that three different clusters were formed by the mammalian, avian and fish CD8α sequences. The thymus was the tissue with the highest CD8α expression, followed by gut, gills, peripheral blood leukocytes and spleen. Lower CD8α mRNA levels were found in head kidney, liver and brain. It was possible to create a partial 3D model using the human and mouse structures as template. The CD8α 11–120 amino acid region was taken into consideration and the best obtained 3D model shows the presence of ten β-strands, involving about 50% of the sequence. The global structure was defined as an immunoglobulin-like β-sandwich made of two anti-parallel sheets. Two cysteines were present in this region and they were at a suitable distance to form an S S bond as seen in the template human and mouse structures.

Bánkúti 1201--an old Hungarian wheat variety with special storage protein composition.

Bánkúti 1201, an old Hungarian wheat variety with special quality traits, was analysed to determine the relationships between its storage protein composition and superior quality-attributes for breadmaking. Based on the storage protein composition, the variety appears to have the nature of a population, containing several genotypes with different gluten protein alleles. Using molecular markers, a new mutant x-type HMW glutenin allele was identified, containing an extra cysteine residue and showing a moderate, positive-effect on gluten properties. In lines possessing subunits Bx7+By8 the overexpression of the Bx-type subunit could be detected, resulting in a higher unextractable polymeric protein (UPP) content and increased dough strength. It was found that the presence or absence of subunit Bx7 has an equilibrating effect on the dough extensibility, which is generally characteristic of the Bánkúti 1201 population. The complex good bread-making quality of the variety, which has strong but highly extensible dough, is probably due to the balance between lines which express subunit Bx7 and those which do not.

Five disulfide bridges stabilize a hevein-type antimicrobial peptide from the bark of spindle tree ( Euonymus europaeus L.)

A small 45 amino acid residue antifungal polypeptide was isolated from the bark of spindle tree ( Euonymus europaeus L.). Though the primary structure of this so-called E. europaeus chitin-binding protein or Ee-CBP is highly similar to the hevein domain, it distinguishes itself from most previously identified hevein-type antimicrobial peptides (AMP) by the presence of two extra cysteine residues that form an extra disulfide bond. Due to these five disulfide bonds Ee-CBP is a remarkably stable protein. Agar diffusion and microtiterplate assays demonstrated that Ee-CBP is a potent antimicrobial protein. IC 50-values as low as 1 μg/ml were observed for the fungus Botrytis cinerea. Comparative assays further demonstrated that Ee-CBP is a stronger inhibitor of fungal growth than Ac-AMP2 from Amaranthus caudatus seeds, which is considered one of the most potent antifungal hevein-type plant proteins.

Analysis of two IgM isotypes in Atlantic salmon and brown trout

Atlantic salmon ( Salmo salar) possesses two distinct subpopulations of polymeric IgM which are separable by anion exchange chromatography. Consistent with this finding there are two isotypic IgM heavy chain genes, CμA and CμB, in the genome of this species, presumably as a result of ancestral tetraploidy. In the present study it was shown that IgM of brown trout ( Salmo trutta) is also separated into two subpopulations by anion exchange chromatography, while IgM of rainbow trout ( Oncorhynchus mykiss) and Arctic char ( Salvelinus alpinus) are eluted in one peak. Molecular cloning of IgM heavy chain cDNAs from brown trout revealed messages of two distinct constant region genes, named CμA and CμB. As deduced from the translated cDNA sequences (and in agreement with isoelectric focusing of the corresponding proteins) the mean pI values of the heavy chains in brown trout differ with only 0.14 units, in comparison to a 0.67 unit difference in salmon. Based on the present sequence analysis we suggest that an additional cysteine near the C-terminus of CμB is critical in relation to the fractionation of IgM by anion exchange chromatography, for example by altering the overall structure of the IgM polymer and the exposure of charged residues. Most likely, the Cμ subvariant with the characteristic extra cysteine residue arose in the ancestor of Atlantic salmon and brown trout, i.e. after the three genera Salmo, Oncorhynchus and Salvelinus radiated.

Conservation of the gene structure and membrane-targeting signals of germ cell-specific lamin LIII in amphibians and fish

Targeting of nuclear lamins to the inner nuclear membrane requires CaaX motif-dependent posttranslational isoprenylation and carboxyl methylation. We previously have shown that two variants of lamin LIII (i.e., LIII and LIIIb) in amphibian oocytes are generated by alternative splicing and differ greatly in their membrane association. An extra cysteine residue (as a potential palmitoylation site) and a basic cluster in conjunction with the CaaX motif function as secondary targeting signals responsible for stable membrane association of lamin LIIIb. cDNA sequencing and genomic analysis of the zebrafish Danio rerio lamin LIII uncovers a remarkable conservation of the genomic organization and of the two secondary membrane-targeting signals in amphibians and fish. The expression pattern of lamin LIII genes is also conserved between amphibians and fish. Danio lamin LIII is expressed in diplotene oocytes. It is absent from male germ cells but is expressed in Sertoli cells of the testis. In addition, we provide sequence information of the entire coding sequence of zebrafish lamin A, which allows comparison of all major lamins from representatives of the four classes of vertebrates.

Characterisation of high- and low-molecular weight glutenin subunits associated to the D genome of Aegilops tauschii in a collection of synthetic hexaploid wheats

Synthetic hexaploid wheats (2n=6x=42, AABBDD) involving genomes from Triticum turgidum (2n= 4x=28, AABB) and Aegilops tauschii (2n=2x=14, DD) have been produced as a means for introducing desirable characteristics into bread wheat. In the present work we describe the genetic variability present at the Glu-D t 1 and Glu-D t 3 loci, encoding high- (HMW) and low-molecular-weight (LMW) glutenin subunits respectively, derived from Ae. tauschii, using electrophoretic and chromatographic methods, in a collection of synthetic hexaploid wheats. A wide variation both in mobility and surface hydrophobicity of HMW glutenin subunits was observed between different accessions of Ae. tauschii used in the production of the synthetic hexaploids. A combination of electrophoretic and chromatographic methods improves the identification of HMW glutenin subunits; in fact subunits with identical apparent mobility were revealed to have a different surface hydrophobicity by reversed-phase high performance liquid chromatography. None of the Dx5t subunits present in Ae. tauschii showed the presence of the extra cysteine residue found in the HMW glutenin subunit Dx5 of Triticum aestivum, as revealed by selective amplification with polymerase chain reaction (PCR). The wide variability and the high number of subunits encoded by the Glu-D t 3 locus suggests that Ae. tauschii may be a rich source for enhancing the genetic variability of glutenin subunits in bread wheat and improving bread-making properties.

Isolation of a cDNA encoding a putative SPARC from the brine shrimp, Artemiafranciscana

SPARC (Secreted protein, acidic, rich in cysteine) is an extracellular matrix-associated and anti-adhesive glycoprotein extensively studied in vertebrates. Its presence among invertebrates has been reported in nematodes and flies. We cloned a cDNA containing a complete open reading frame for SPARC from the brine shrimp, Artemiafranciscana. The amino acid sequence identity between the Artemia and the fly SPARCs was 55%, whereas that of the Artemia and the nematode proteins was 45%. Artemia and vertebrates exhibited a sequence identity of 30% in the predicted aa sequences. The SPARC consisted of four domains commonly found among reported SPARCs. The protein comprised 291 amino acids, having a signal peptide, a follistatin-like domain, one N-glycosylation site and one calcium-binding EF-hand motif. Fourteen cysteine residues conserved among all the secreted forms of SPARCs were present in the Artemia SPARC, and four extra cysteine residues were also found in it. The extra residues were conserved among SPARCs of the arthropods and the nematode. Phylogenetic analyses showed that the sequences of SPARCs were grouped into those of vertebrates and invertebrates. Though the structural organization of SPARC was conserved among all the species studied, SPARC within a group was highly conserved within that group, but divergent between the two. Northern blots revealed the presence of a 1.1 kb mRNA, which was faintly expressed in embryos and considerably detected in prenauplii and nauplii. The isolation of a SPARC cDNA from Artemiafranciscana provides intriguing features of the divergent protein, SPARC.

MOUSE T-CELL ANTIGEN Rt6.1 HAS THIOL-DEPENDENT NAD GLYCOHYDROLASE ACTIVITY

Mouse Rt6.1 and Rt6.2, homologues of rat T-cell RT6 antigens, catalyze arginine-specific ADP-ribosylation. Without an added ADP-ribose acceptor, Rt6.2 shows NAD glycohydrolase (NADase) activity. However, Rt6.1 has been reported to be primarily an ADP-ribosyltransferase, but not an NADase. In the present study, we obtained evidence that recombinant Rt6.1 catalyzes NAD glycohydrolysis but only in the presence of DTT. The NADase activity of Rt6.1 observed in the presence of DTT was completely inhibited by N-ethylmaleimide (NEM). Native Rt6.1 antigen, immunoprecipitated from BALB/c mouse splenocytes with polyclonal antibodies generated against recombinant RT6.1, also exhibited NADase activity in the presence of DTT. Compared with Rt6.2, Rt6.1 has two extra cysteine residues at positions 80 and 201. When Cys-80 and Cys-201 in Rt6.1 were replaced with the corresponding residues of Rt6.2, serine and phenylalanine, respectively, Rt6.1 catalyzed the NADase reaction even in the absence of DTT. Conversely, replacing Ser-80 and Phe-201 in Rt6.2 with cysteines, as in Rt6.1, converted the thiol-independent Rt6.2 NADase to a thiol-dependent enzyme. Kinetic study of the NADase reaction revealed that the affinity of Rt6.1 for NAD and the rate of catalysis increased in the presence of DTT. Moreover, the NADase activity of Rt6.1 expressed on COS-7 cells was stimulated by culture supernatant from activated mouse macrophages, even in the absence of DTT. From these observations, we conclude that t!he Rt6.1 antigen has thiol-dependent NADase activity, and that Cys-80 and Cys-201 confer thiol sensitivity to Rt6.1 NADase. Our results also suggest that upon the interaction of T-cells expressing Rt6.1 with activated macrophages, the NADase activity of the antigen will be stimulated.

Mouse T-cell antigen rt6.1 has thiol-dependent NAD glycohydrolase activity.

Mouse Rt6.1 and Rt6.2, homologues of rat T-cell RT6 antigens, catalyze arginine-specific ADP-ribosylation. Without an added ADP-ribose acceptor, Rt6.2 shows NAD glycohydrolase (NADase) activity. However, Rt6.1 has been reported to be primarily an ADP-ribosyltransferase, but not an NADase. In the present study, we obtained evidence that recombinant Rt6.1 catalyzes NAD glycohydrolysis but only in the presence of DTT. The NADase activity of Rt6.1 observed in the presence of DTT was completely inhibited by N-ethylmaleimide (NEM). Native Rt6.1 antigen, immunoprecipitated from BALB/c mouse splenocytes with polyclonal antibodies generated against recombinant RT6.1, also exhibited NADase activity in the presence of DTT. Compared with Rt6.2, Rt6.1 has two extra cysteine residues at positions 80 and 201. When Cys-80 and Cys-201 in Rt6.1 were replaced with the corresponding residues of Rt6.2, serine and phenylalanine, respectively, Rt6.1 catalyzed the NADase reaction even in the absence of DTT. Conversely, replacing Ser-80 and Phe-201 in Rt6.2 with cysteines, as in Rt6.1, converted the thiol-independent Rt6.2 NADase to a thiol-dependent enzyme. Kinetic study of the NADase reaction revealed that the affinity of Rt6.1 for NAD and the rate of catalysis increased in the presence of DTT. Moreover, the NADase activity of Rt6.1 expressed on COS-7 cells was stimulated by culture supernatant from activated mouse macrophages, even in the absence of DTT. From these observations, we conclude that t!he Rt6.1 antigen has thiol-dependent NADase activity, and that Cys-80 and Cys-201 confer thiol sensitivity to Rt6.1 NADase. Our results also suggest that upon the interaction of T-cells expressing Rt6.1 with activated macrophages, the NADase activity of the antigen will be stimulated.

A Novel Tissue Inhibitor of Metalloproteinases-3 Mutation Reveals a Common Molecular Phenotype in Sorsby's Fundus Dystrophy

A Novel Tissue Inhibitor of Metalloproteinases-3 Mutation Reveals a Common Molecular Phenotype in Sorsby's Fundus Dystrophy