Showing papers on "Protein primary structure published in 2006"
TL;DR: Carohydrate compositional as well as methylation analysis indicated that Asn29and Asn56 bear exclusively complex‐type oligosaccharide structures that are almost quantitatively α1‐6 fucosylated at the proximal N‐acetylglucosamine; ∼70% of these molecules contain a bisecting N‐ acetylglUCosamine.
Abstract: beta-Trace protein from pooled human CSF was purified to homogeneity. An apparent molecular mass of 23-29 kDa was determined for the polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal sequencing of the polypeptide yielded the unique amino acid sequence APEAQVSVQPNFQQDKFLGRWFSA. Alignment of amino acid sequences obtained from tryptic peptides with the sequence previously deduced from a cDNA clone isolated by other investigators allowed the identification of beta-trace protein as prostaglandin D synthase [prostaglandin-H2 D-isomerase; (5Z, 13E)-(15S)-9 alpha, 11 alpha-epidioxy-15-hydroxyprosta-5,13-dienoate D-isomerase; EC 5.3.99.2]. A conservative amino acid exchange (Thr instead of Ser) was detected at amino acid position 154 of the beta-trace polypeptide chain in the corresponding tryptic peptide. The two N-glycosylation sites of the polypeptide were shown to be almost quantitatively occupied by carbohydrate. Carbohydrate compositional as well as methylation analysis indicated that Asn29 and Asn56 bear exclusively complex-type oligosaccharide structures (partially sialylated with alpha 2-3- and/or alpha 2-6-linked N-acetylneuraminic acid) that are almost quantitatively alpha 1-6 fucosylated at the proximal N-acetylglucosamine; approximately 70% of these molecules contain a bisecting N-acetylglucosamine. Agalacto structures as well as those with a peripheral fucose are also present.
182 citations
TL;DR: A novel 40-residue antimicrobial peptide, aurelin, exhibiting activity against Gram-positive and Gram-negative bacteria, was purified from the mesoglea of a scyphoid jellyfish Aurelia aurita by preparative gel electrophoresis and RP-HPLC.
169 citations
TL;DR: The overall folded protein conformation is globular with an exposed peptide loop toward the carboxy-end of the primary structure, with an asymmetric charge distribution, with high density of basic residues concentrated on one side of the molecule and of acidic residues on the opposite side.
117 citations
TL;DR: The relationship between the structure of Yaa-3 and HSP47 binding using synthetic collagenous peptides and a motif search against human protein database predicted candidate clients for this molecular chaperone indicated that not all collagen family proteins require the chaperoning by H SP47.
70 citations
TL;DR: Phylogenetic analysis, primary structure alignments and higher order structural predictions all suggest that the P. furiosus protein is related to proteins within the broad superfamily of ferritin-like di-iron carboxylate proteins, which contributes to its service as a multifunctional antioxidant.
52 citations
TL;DR: This work describes a novel antimicrobial peptide isolated from the salivary glands of the hard tick, Ixodes sinensis, that lacks cysteine in its primary structure and exerted its antimicrobial activities against bacteria and fungi.
46 citations
TL;DR: The entire primary structure of AT is substituted with alanine or cysteine to identify those amino acids that comprise functional domains involved in receptor binding, oligomerization, and pore formation and revealed that receptor binding is restricted to domain 1 of the AT structure.
Abstract: Alpha toxin (AT) is the major virulence factor of Clostridium septicum that is a proteolytically activated pore-forming toxin that belongs to the aerolysin-like family of toxins. AT is predicted to be a three-domain molecule on the basis of its functional and sequence similarity with aerolysin, for which the crystal structure has been determined. In this study, we have substituted the entire primary structure of AT with alanine or cysteine to identify those amino acids that comprise functional domains involved in receptor binding, oligomerization, and pore formation. These studies revealed that receptor binding is restricted to domain 1 of the AT structure, whereas domains 1 and 3 are involved in oligomerization. These studies also revealed the presence of a putative functional region of AT proximal to the receptor-binding domain but distal from the pore-forming domain that is proposed to regulate the insertion of the transmembrane β-hairpin of the prepore oligomer.
44 citations
TL;DR: It was concluded that systematic alignment of sequences of related virus strains can be used to test hypotheses derived from molecular studies of individual model viruses and to generate ideas for future studies on virus structure and replication.
Abstract: An alignment was made of the deduced amino acid sequences of the entire capsid protein VP1 of all human rhinovirus (HRV) prototype strains to examine conserved motifs in the primary structure. A set of previously proposed crucially important amino acids in the footprints of the two known receptor molecules was not conserved in a receptor group-specific way. In contrast, VP1 and VP3 amino acids in the minor receptor-group strains corresponding to most of the predicted ICAM-1 footprint definitely differed from those of the ICAM-1-using major receptor-group strains. Previous antiviral-sensitivity classification showed an almost-complete agreement with the species classification and a fair correlation with amino acids aligning in the antiviral pocket. It was concluded that systematic alignment of sequences of related virus strains can be used to test hypotheses derived from molecular studies of individual model viruses and to generate ideas for future studies on virus structure and replication.
42 citations
TL;DR: This structure-activity study provides increased insight into active site complexity and catalytic mechanism of 11beta-HSD1, useful for further inhibitor design.
42 citations
TL;DR: The formation of many products, including hydroxylation, is inhibited by proximity to negative charges, while the formation of other products showed more complicated responses to changing primary sequence, and the relative amounts of certain oxidation products can be heavily influenced by the primary structure of the surrounding peptide.
Abstract: Hydroxyl radical surface mapping is a useful tool for investigating protein structure and folding. The rate of protein side-chain oxidation by the hydroxyl radical is known to be affected primarily by the chemical reactivity of the side chain and the accessibility of the reactive site to the radical. Efforts have been made to determine the inherent rate of stable product formation of each amino acid side chain, so that the rate of oxidation of an amino acid can be used to accurately estimate the average solvent accessibility of the amino acid side chain in the folded protein. However, the effects of nearby primary sequence on peptide oxidation have not been studied. Here, we examine the amounts of various oxidation products of a small peptide consisting of one leucine and one aspartic acid separated by zero to five glycine residues, as well as with modification of the N- and C-terminus. We find that the relative amounts of certain oxidation products can be heavily influenced by the primary structure of the surrounding peptide. The formation of many products, including hydroxylation, is inhibited by proximity to negative charges, while the formation of other products showed more complicated responses to changing primary sequence.
34 citations
TL;DR: In the European Molecular Biology data bank, CP is reported as the first member of the CP family; this is the first example of an set of secreted fungal proteins whose primary structure is very similar to proteins of the hydrophobin family.
Abstract: The ascomycete Ceratocystis fimbriata, the causal agent of "canker stain disease," secretes a protein of 12.4 kDa that elicits phytoalexin synthesis and plant cell death. This protein, named cerato-platanin (CP), is also located in the cell walls of ascospores, hyphae, and conidia; it contains four cysteines (S-S bridged) and is moderately hydrophobic. The cp gene consists of a single exon and has 42 bp codifying for a signal peptide of 14 residues. The recombinant protein was obtained by cloning the cp gene of the mature protein in Escherichia coli (BL21), and a refolding step was needed to achieve the native active form. In the European Molecular Biology data bank, CP is reported as the first member of the CP family; this is the first example of an set of secreted fungal proteins whose primary structure is very similar. Nonetheless, the data also revealed some structural and functional features that make CP similar to proteins of the hydrophobin family.
TL;DR: The multifunctional behavior of integrins based on their modification potential at hydroxyl groups of amino acids as a source of interplay is described.
Abstract: Protein functions are determined by their three-dimensional structures and the folded 3-D structure is in turn governed by the primary structure and post-translational modifications the protein undergoes during synthesis and transport. Defining protein functions in vivo in the cellular and extracellular environments is made very difficult in the presence of other molecules. However, the modifications taking place during and after protein folding are determined by the modification potential of amino acids and not by the primary structure or sequence. These post-translational modifications, like phosphorylation and O-linked N-acetylglucosamine (O-GlcNAc) modifications, are dynamic and result in temporary conformational changes that regulate many functions of the protein. Computer-assisted studies can help determining protein functions by assessing the modification potentials of a given protein. Integrins are important membrane receptors involved in bi-directional (outside-in and inside-out) signaling events. The beta3 integrin family, including, alpha(IIb)beta3 and alpha(v)beta3, has been studied for its role in platelet aggregation during clot formation and clot retraction based on hydroxyl group modification by phosphate and GlcNAc on Ser, Thr, or Tyr and their interplay on Ser and Thr in the cytoplasmic domain of the beta3 subunit. An antagonistic role of phosphate and GlcNAc interplay at Thr758 for controlling both inside-out and outside-in signaling events is proposed. Additionally, interplay of GlcNAc and phosphate at Ser752 has been proposed to control activation and inactivation of integrin-associated Src kinases. This study describes the multifunctional behavior of integrins based on their modification potential at hydroxyl groups of amino acids as a source of interplay.
TL;DR: This study provides evidence that GCG is the LECT1 gene product, while neither a previously described LECT2 gene nor a functional Lect2 gene product was found in the specimen under investigation.
Abstract: The ancestral galectin from the sponge Geodia cydonium (GCG) is classified on a structural basis to the prototype subfamily, whereas its carbohydrate-binding specificity is related to that of the mammalian chimera-type galectin-3. This dual coordination reveals GCG as a potential precursor of the later evolved galectin subfamilies, which is reflected in the primary structure of the protein. This study provides evidence that GCG is the LECT1 gene product, while neither a previously described LECT2 gene nor a functional LECT2 gene product was found in the specimen under investigation. The electrophoretically separated protein isomers with apparent molecular masses of 13, 15, and 16 kDa correspond to variants of the LECT1 protein-exhibiting peptide sequence polymorphisms that concern critical positions of the carbohydrate recognition domain (13 kDa: Leu51, Asn55, His130, Gly137; 15 kDa: Ser51, Asn55, Asn130, Gly137; 16 kDa: Ser51, Tyr55, Asn130, Glu137). Four residues, highly conserved in the galectin family, are substituted. None of the residues claimed to be involved in interactions with GalNAcalpha1-3 moieties at an extended binding subsite of galectin-3 was identified in the corresponding positions of GCG. Apparently, the substitutions do not confer distinct binding characteristics to the GCG variants as evidenced by binding studies with a recombinantly expressed 15-kDa isoform. The natural isoforms as well as the recombinant 15-kDa isoform oligomerize by the formation of non-covalent heteromeric or homomeric complexes. A phosphorylation of the galectin was confirmed neither by mass spectrometry nor by alkaline phosphatase treatment combined with isoelectric focusing.
TL;DR: A cDNA encoding an open reading frame of 199 amino acids corresponding to a type II peroxiredoxin from Pisum sativum with its transit peptide was isolated by RT-PCR and molecular replacement using a theoretical model predicted from the primary structure as a search model confirmed the presence of six molecules in the unit cell as expected from the Matthews coefficient.
Abstract: A cDNA encoding an open reading frame of 199 amino acids corresponding to a type II peroxiredoxin from Pisum sativum with its transit peptide was isolated by RT-PCR. The 171-amino-acid mature protein (estimated molecular weight 18.6 kDa) was cloned into the pET3d vector and overexpressed in Escherichia coli. The recombinant protein was purified and crystallized by the hanging-drop vapour-diffusion technique. A full data set (98.2% completeness) was collected using a rotating-anode generator to a resolution of 2.8 A from a single crystal flash-cooled at 100 K. X-ray data revealed that the protein crystallizes in space group P1, with unit-cell parameters a = 61.88, b = 66.40, c = 77.23 A, α = 102.90, β = 104.40, γ = 99.07°, and molecular replacement using a theoretical model predicted from the primary structure as a search model confirmed the presence of six molecules in the unit cell as expected from the Matthews coefficient. Refinement of the structure is in progress.
TL;DR: The identification and characterization of Dusty is described, a single-copy gene that arose in metazoan evolution and encodes a putative dual Ser/Thr and Tyr protein kinase with unique structural features that may play important functional roles in the brain and other tissues of vertebrates.
TL;DR: In contrast to prevailing expectations, (Gly–Asp)n-type sequence motifs exist in MPSP, demanding a revision of previous theories of protein–mineral interactions, and the cDNA sequence of myostracum is elucidated for the first time.
Abstract: Soluble protein (MPSP, myostracal prism soluble protein) obtained from
myostracum in oyster shell (Crassostrea gigas) was characterized using biochemical and molecular biological techniques. From an analysis of secondary protein structure, it was shown that β-structure was predominant in MPSP. And via in vitro assays, the relation of MPSP to biomineral phase and morphology was studied. SDS-PAGE revealed one major protein band of 20 kDa. An amino acid sequence of 160 amino acids was deduced for myostracum by characterization of the complementary DNA encoding the protein. The deduced protein was composed of a high proportion of Gly and Asp, typifying a calcium-binding protein for shell formation, and a relatively high proportion of Val, Ala and Ile, typifying an adhesive protein. In contrast to prevailing expectations, (Gly–Asp)n-type sequence motifs exist in MPSP, demanding a revision of previous theories of protein–mineral interactions. The cDNA sequence of myostracum is elucidated for the first time.
TL;DR: Using RT-PCR analysis, it was found that the B. mori PP-BP gene was expressed almost exclusively in the hemolymph, and real-time quantitative PCR analysis indicated thatThe B.moriPP-BP is related to the cellular immune response after BmNPV invades the hemologic immune reaction.
TL;DR: It is shown that, although BS-producing Streptomyces morookaensis also produces a PM-inactivating enzyme, it catalyzes the hydrolysis of an amide linkage between the aminonucleoside and O-methyl-L-tyrosine moiety of PM, suggesting that PMH is an aminopeptidase classified into the POP family.
TL;DR: The potential ability of nuclear protein ACR7 to bind Gln and the possibility of the protein acting as a Gln sensor in rice leaves is discussed.
Abstract: The regulatory ACT domains serve as amino acid-binding sites in some amino acid metabolic enzymes and transcriptional regulators in bacteria. To elucidate the molecular roles of the glutamine (Gln)-sensing system in nitrogen (N) metabolism in plants, we isolated six genes encoding ACT domain repeat proteins (ACR1, and ACR5-ACR9) from rice (Oryza sativa L.) using genomic information on the primary structure composed of four copies of the domain homologous to those of bacterial Gln sensor GLND. Since expression of ACR7 was the most abundant of the six ACR orthologous genes, we focused on this ACR in the current study. Gene products of ACR7 were most abundant in young developing leaf blades of rice, and ACR7 protein is specifically localized in the nucleus of the parenchyma cells of phloem and xylem in the vascular bundles. A yeast two-hybrid screen identified a small heat stress protein (HSP18.0-CII) as a protein interacting with ACR7. Transient expression analysis of HSP18.0-CII:sGFP in cultured rice cells, followed by co-immunoprecipitation, suggests that the nuclear ACR7 indeed interacted with nucleocytoplasmic HSP18.0-CII in vivo. The potential ability of nuclear protein ACR7 to bind Gln and the possibility of the protein acting as a Gln sensor in rice leaves is discussed.
TL;DR: High sequence coverage resulting from multiple proteolytic cleavage, MALDI‐TOF/TOF, Nano‐HPLC‐ESI‐MS/MS and de novo sequencing completed unambiguous analysis of SLP‐2 primary structure of ≈90% of sequence coverage.
Abstract: Stomatin-like protein 2 (SLP-2) (syn.: EPB72-like 2 [NP_038470], HSPC108 [AAF29073]), a protein of unknown function, has been described in several tissues and cells but its primary structure is still not completely elucidated. Moreover, sequence conflicts appear in several databases. It was the aim of the study to further describe SLP-2 primary sequence and to solve existing sequence conflicts. For this purpose a protein extract was run on two-dimensional gel electrophoresis and SLP-2 was identified by MALDI-TOF/TOF. SLP-2 was digested with trypsin, chymotrypsin, Lys-C, and de novo sequencing studies as well as Nano-HPLC-ESI-MS/MS analysis were carried out. By the use of several proteases sequence coverage of 90% was obtained but the N-terminal 34 amino acids harbouring database conflict 1 were not covered. The presence of Leucine 129 (sequence conflict 2) and Alanine 202 (sequence conflict 3) was verified by three independent approaches. High sequence coverage resulting from multiple proteolytic cleavage, MALDI-TOF/TOF, Nano-HPLC-ESI-MS/MS and de novo sequencing completed unambiguous analysis of SLP-2 primary structure of ≈90% of sequence coverage. In addition, methodology used was able to solve so far pending sequence conflicts in databases and literature. SLP-2 is a high abundance protein in several tissues and cells and may play an important biological role and therefore characterization of its primary structure is of importance. Proteins 2006. © 2006 Wiley-Liss, Inc.
TL;DR: To better understand the role of SPARC in mineralizing systems, isolated SPARC from developing pig teeth, deduced its primary structure from the cDNA sequence, and determined its quaternary structure by homology modelling with reference to human SPARC crystal structures.
Abstract: Genes encoding the major enamel matrix proteins and non-collagenous proteins of bone and dentin are members of the secretory calcium-binding phosphoprotein (SCPP) family, which originated from ancestral SPARC (secreted protein, acidic and rich in cysteine; BM-40/osteonectin). To better understand the role of SPARC in mineralizing systems, we isolated SPARC from developing pig teeth, deduced its primary structure from the cDNA sequence, and determined its quaternary structure by homology modelling with reference to human SPARC crystal structures. The guanidine/EDTA extract from porcine dentin was fractionated by anion-exchange and size-exclusion chromatography. Stains-all positive bands at 38 and 35 kDa gave the N-terminal sequences APQQEALPDETEV and DFEKNYNMYIFPV, which corresponded to the SPARC N terminus and an internal region of the protein. Porcine SPARC contains 300 amino acids, including the 17-amino acid signal peptide, and shares 96.2% amino acid sequence identity with human SPARC. Without post-translational modifications, the 283-amino acid secreted protein has a molecular mass of 32.3 kDa. The three-dimensional model revealed that porcine SPARC contains a single N-linked glycosylation at N113, seven intramolecular disulfide bridges, and assembles into dimers. SPARC is composed of three structural/functional domains: an acidic Ca 2+ -binding, a follistatin-like, and an extracellular calcium-binding domain.
TL;DR: Most thoroughly investigated snake venom factor X activators are from Vipera russelli and Vipera lebetina and VLFXA, which consist of a heavy chain and two C-type lectin–like light chains that are held together by disulfide bonds.
Abstract: A variety of factor X activators have been detected in snake venoms. About 10 activators have been isolated from Viperidae, Crotalidae, and Elapidae venoms. Viperidae and Crotalidae venom activators are mainly metalloproteases. Only two factor X activators are characterized from Elapidae venoms, both belonging to serine proteases. Most thoroughly investigated snake venom factor X activators are from Vipera russelli (now renamed Daboia russelli; RVV-X) and Vipera lebetina (now renamed Macrovipera lebetina; VLFXA). VLFXA is cloned and sequenced, and its primary structure is deduced from the cDNA sequence. Both activators consist of a heavy chain and two C-type lectin–like light chains that are held together by disulfide bonds. Primary stuctures of the heavy chain and one light chain are similar for both activators. The primary structure of the second light chain is not detected in RVV-X. Heavy chains of RVV-X and VLFXA contain metalloprotease and disintegrin-like and cysteine-rich domains. All chains of VLF...
TL;DR: The findings reported here make possible the design of a B3GTL "knock-out" mouse, provide a framework for analyzing the regulation of the gene, and provide an extensive catalog of tissues in which this novel protein acts.
Abstract: Glycosylation of proteins and lipids is important in cellular communication and maintenance of tissues. B3GTL (β3-glycosyltransferase-like) is a novel glycosyltransferase that is found in multicellular animals ranging from mammals to insects and nematodes. The aim of this work was to identify and characterize the B3GTL gene in the mouse and to study its expression in various tissues. The murine gene codes for a protein which shares 84% amino acid sequence identity with its human ortholog, and contains all the primary structural features that characterize B3GTL proteins. The murine and human B3GTL genes share an identical exon/intron organization, and both genes utilize multiple polyadenylation signals. Their promoter regions show extensive conservation, implying that the two genes also share regulatory similarities. This notion was reinforced by Northern hybridization analysis of mouse tissues, which showed the tissue distribution of B3GTL mRNA to be similar to that previously found in human tissues, with...
TL;DR: The structural and functional characteristics suggest that AvPDI is another member of the PDI protein family and represents the first more closely characterized PDI in the ticks.
TL;DR: It is shown that SLA I cell-surface expression in the pig epithelial cell line LLC-PK1 is relatively unaffected by expression of ICP47, and it is unable to demonstrate that this imparted an apparent resistance to the effects of I CP47 onSLA I surface expression.
Abstract: The transporter associated with antigen processing (TAP) is a heterodimer composed of TAP1 and TAP2 subunits that belong to the ATP-binding cassette family of transporters. TAP translocates small peptides (usually 8- to 12-amino-acid-long) from the cytosol to the endoplasmic reticulum for subsequent loading onto the major histocompatibility complex (MHC) class I molecules. The translocated peptides are required for the stable cell surface expression of MHC class I molecules. Virus-encoded proteins, which inhibit TAP activity, include ICP47 from herpes simplex virus and US6 from human cytomegalovirus. We have previously shown that ICP47 downregulated porcine MHC class I [swine leukocyte Ag class I (SLA I)] cell-surface expression in the pig epithelial cell line PK(15). Here we show that SLA I cell-surface expression in the pig epithelial cell line LLC-PK1 is relatively unaffected by expression of ICP47. Anticipating that this might be due to differences in the primary structure of TAP1 or TAP2 expressed by these two cell lines, cDNAs from PK(15) and LLC-PK1 encoding the complete open reading frames of porcine TAP1 and TAP2 were cloned and sequenced. Porcine TAP1 and TAP2 exhibited 80% amino acid identity with their human orthologs. Two splice variants of TAP1 were found. In LLC-PK1 cells, an alternatively spliced TAP1 transcript was detected, which was predicted to encode a protein with nine fewer amino acids. While the deleted amino acids may be in close proximity to the putative peptide/ICP47-binding site, we were unable to demonstrate that this imparted an apparent resistance to the effects of ICP47 on SLA I surface expression.
TL;DR: The enzyme responsible for the hydrolysis of phosphonoacetic acid, a non-biogenic C-P compound, was purified to electrophoretic homogeneity from a wild-type strain of Penicillium oxalicum and showed a high degree of similarity with the few sequences available to date for the bacterial enzyme.
TL;DR: In this paper, a potential amphipathic alpha-helix in the N-terminal ATPase domain of heat shock proteins was identified, which shares 89% similarity to the experimentally determined calmodulin-binding domain from mouse, Hsc70, and is conserved in nearly half of the 113 members of the HSP70 family investigated.
Abstract: Yeast soluble proteins were fractionated by calmodulin-agarose affinity chromatography and the Ca2+/calmodulin-binding proteins were analyzed by SDS-PAGE. One prominent protein of 66 kDa was excised from the gel, digested with trypsin and the masses of the resultant fragments were determined by MALDI/MS. Twenty-one of 38 monoisotopic peptide masses obtained after tryptic digestion were matched to the heat shock protein Ssb1/Hsp75, covering 37% of its sequence. Computational analysis of the primary structure of Ssb1/Hsp75 identified a unique potential amphipathic alpha-helix in its N-terminal ATPase domain with features of target regions for Ca2+/calmodulin binding. This region, which shares 89% similarity to the experimentally determined calmodulin-binding domain from mouse, Hsc70, is conserved in near half of the 113 members of the HSP70 family investigated, from yeast to plant and animals. Based on the sequence of this region, phylogenetic analysis grouped the HSP70s in three distinct branches. Two of them comprise the non-calmodulin binding Hsp70s BIP/GR78, a subfamily of eukaryotic HSP70 localized in the endoplasmic reticulum, and DnaK, a subfamily of prokaryotic HSP70. A third heterogeneous group is formed by eukaryotic cytosolic HSP70s containing the new calmodulin-binding motif and other cytosolic HSP70s whose sequences do not conform to those conserved motif, indicating that not all eukaryotic cytosolic Hsp70s are target for calmodulin regulation. Furthermore, the calmodulin-binding domain found in eukaryotic HSP70s is also the target for binding of Bag-1 - an enhancer of ADP/ATP exchange activity of Hsp70s. A model in which calmodulin displaces Bag-1 and modulates Ssb1/Hsp75 chaperone activity is discussed.
TL;DR: This work has demonstrated p53-binding activity by an additional region, and confirmed previous results showing that two regions of BRCA1 protein bind p53 in vitro, and predicted five p 53-binding sites based on sequence analysis.
TL;DR: The results demonstrate that this 1200K-protein is a connectin-like elastic protein and includes specific PEVK-like fragment, which is suggested to play a major role in maintaining the structure of oblique muscle in invertebrates.
Abstract: Vertebrate striated muscle contains the giant elastic protein connectin that maintains the position of the A-band at the center of the sarcomere during repeated muscular contraction and relaxation. Connectin-like molecules may perform conserved functions in vertebrate and invertebrate striated and oblique muscles, although less is known about the structure of invertebrate connectins at present. The protein that maintains such a structure is present not only in vertebrate striated muscle, but also in invertebrate striated and oblique muscle. In the present study, we analyzed the partial primary structure of a 1200K-protein, which is a connectin-like protein that is expressed in Neanthes sp. body wall muscle that is in turn composed of oblique muscle. Antibody screening of a cDNA library of Neanthes sp. body wall muscle identified two different clones. Both clones coded for a sequence predominantly comprised of the four amino acids proline (P), glutamate (E), valine (V) and lysine (K). One clone included a PEVK-like repeat sequence flanked by an Ig domain, while the other clone comprised a distinct 14 amino acid repeat rich in PEVK residues, flanked by a non-repetitive unique sequence. The PEVK region is found in vertebrate connectin and is thought to generate elasticity and be responsible for passive tension of the muscle. The antibodies produced against a portion of each clone both reacted with bands corresponding to 1200 kDa present in Neanthes sp. body wall muscle. Therefore, our results demonstrate that this 1200K-protein is a connectin-like elastic protein and includes specific PEVK-like fragment. We suggest that this 1200K-protein plays a major role in maintaining the structure of oblique muscle in invertebrates.
TL;DR: Preliminary experiments demonstrate that the operon is expressed under the control of its own promoter in Escherichia coli, providing evidence that additional proteins have to be involved in the maturation process.
Abstract: The complete primary structure of the hyn-region in the genome of Desulfovibrio vulgaris Miyazaki F (DvMF), encoding the [NiFe]-hydrogenase and two maturation proteins has been identified. Besides the formerly reported genes for the large and small subunits, this region comprises genes encoding an endopeptidase (HynC) and a putative chaperone (HynD). The complete genomic region covers 4086 nucleotides including the previously published upstream located promoter region and the sequences of the structural genes. A phylogenetic tree for both maturation proteins shows strongest sequential relationship to the orthologous proteins of Desulfovibrio vulgaris Hildenborough (DvH). Secondary structure prediction for HynC (168 aa, corresponding to a molecular weight of 17.9 kDa) revealed a practically identical arrangement of α-helical and β-strand elements between the orthologous protein HybD from E. coli and allowed a three-dimensional modelling of HynC on the basis of the formerly published structure of HybD. The putative chaperone HynD consists of 83 aa (molecular weight of 9 kDa) and shows 76% homology to DvH HynD. Preliminary experiments demonstrate that the operon is expressed under the control of its own promoter in Escherichia coli, although no further processing could be observed, providing evidence that additional proteins have to be involved in the maturation process. Accession numbers: DQ072852, HynC protein ID AAY90127, HynD protein ID AAY90128.