Amino acid regions of family 45 endoglucanases involved in cotton defibrillation and in resistance to anionic surfactants and oxidizing agents

In the detergent industry, fungal endoglucanases are used to release microfibrils from the surfaces of dyed cellulosic fabrics to enhance color brightness. Family 45 endoglucanase (glycoside hydrolase family 45, GH45) EGL3 from Humicola grisea is more resistant to anionic surfactants and oxidizing agents than family 45 endoglucanase RCE1 from Rhizopus oryzae, while in the present study, a catalytic domain of RCE1 had higher defibrillation activity on dyed cotton fabrics than did that of EGL3. To identify the amino acid regions involved in these properties, we compared the characteristics of RCE1, EGL3, and three chimeric endoglucanases, in which each of the three regions of the catalytic domain of EGL3 was replaced by the corresponding region of the catalytic domain of RCE1. Amino acids in the N-terminal region were involved in resistance to anionic surfactants and oxidizing agents. Furthermore, amino acids in the region adjacent to the N-terminal region were involved in releasing microfibrils and in binding to dyed cotton fabrics, indicating that the binding of the amino acids in this region might be important in the release of microfibrils from dyed cotton fabrics.     

Amino Acid Regions of Family 45 Endoglucanases Involved in Cotton Defibrillation and in Resistance to Anionic Surfactants and Oxidizing Agents

In the detergent industry, fungal endoglucanases are used to release microfibrils from the surfaces of dyed cellulosic fabrics to enhance color brightness. Family 45 endoglucanase (glycoside hydrolase family 45, GH45) EGL3 from Humicola grisea is more resistant to anionic surfactants and oxidizing agents than family 45 endoglucanase RCE1 from Rhizopus oryzae, while in the present study, a catalytic domain of RCE1 had higher defibrillation activity on dyed cotton fabrics than did that of EGL3. To identify the amino acid regions involved in these properties, we compared the characteristics of RCE1, EGL3, and three chimeric endoglucanases, in which each of the three regions of the catalytic domain of EGL3 was replaced by the corresponding region of the catalytic domain of RCE1. Amino acids in the N-terminal region were involved in resistance to anionic surfactants and oxidizing agents. Furthermore, amino acids in the region adjacent to the N-terminal region were involved in releasing microfibrils and in binding to dyed cotton fabrics, indicating that the binding of the amino acids in this region might be important in the release of microfibrils from dyed cotton fabrics.     

Purification and characterization of a new endo-1,4-beta-D-glucanase from Beltraniella portoricensis

A new endoglucanase, designated BCE1, produced by Beltraniella portoricensis, was purified from the culture supernatant. The N-terminal amino acid sequence suggests that BCE1 belongs to family 45 glycoside hydrolase (family 45 endoglucanase). The molecular mass of BCE1 was found to be 40 kDa by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH for the carboxymethyl cellulase (CMCase) activity of BCE1 was 4.5, and the optimum temperature was 55 degrees C. Among family 45 endoglucanases, RCE1 and RCE2 from Rhizopus oryzae, PCE1 from Phycomyces nitens, and EGL3 and EGL4 from Humicola grisea, BCE1 was most resistant to anionic surfactant and oxidizing agent. These results indicate that BCE1 might prove to be a useful enzyme in the detergent industry.     

Purification and characterization of a new family 45 endoglucanase, STCE1, from Staphylotrichum coccosporum and its overproduction in Humicola insolens

In the detergent industry, fungal endoglucanases have been used to release microfibrils (defibrillation) from the surface of dyed cellulosic fabrics to enhance color brightness. Although endoglucanases for laundry use must have various properties, such as a neutral or alkaline optimum pH, resistance to anionic surfactants and oxidizing agents (main components in detergents), and high defibrillation activity, all-purpose endoglucanases have not been obtained yet. As a result of screening of endoglucanases, a new family 45 endoglucanase (family 45 glycoside hydrolase), designated STCE1, was obtained and purified to apparent homogeneity from the culture supernatant of Staphylotrichum coccosporum NBRC 31817. The molecular mass of STCE1 was 49 kDa. The optimum pH for the carboxymethyl cellulase activity of STCE1 was 6.0, and the optimum temperature was 60 degrees C. STCE1 was highly resistant to an anionic surfactant and an oxidizing agent. Furthermore, the defibrillation activities on dyed cotton and lyocell fabrics of STCE1 were higher than those of the other representative endoglucanases tested. These results indicate that STCE1 is an all-purpose enzyme for laundry use. A gene encoding STCE1, designated the stce1 gene, was cloned from S. coccosporum, and the complete sequence was determined. STCE1 consisted of three distinct domains: an N-terminal catalytic domain (family 45), a linker domain, and a C-terminal carbohydrate-binding module (family 1). The amino acid sequences of the catalytic domain of STCE1 were phylogenetically close to those of the family 45 endoglucanases EGL3, EGL4, and EGV from a Humicola sp. Hence, the stce1 gene was transferred into Humicola insolens and expressed. As a result, extremely high levels (0.90 mg protein per ml of culture supernatant, 27% of the total proteins) of the recombinant STCE1 were secreted as a mature form in the culture supernatant.     

Gene duplication event in family 12 glycosyl hydrolase from Phytophthora spp

A total of 18 paralogs of xyloglucan-specific endoglucanases (EGLs) from the glycosyl hydrolase family 12 were identified and characterized in Phytophthora sojae and Phytophthora ramorum. These genes encode predicted extracellular enzymes, with sizes ranging from 189 to 435 amino acid residues, that would be capable of hydrolyzing the xyloglucan component of the host cell wall. In two cases, four and six functional copies of these genes were found in tight succession within a region of 5 and 18 kb, respectively. The overall gene copy number and relative organization appeared well conserved between P. sojae and P. ramorum, with apparent synteny in this region of their respective genomes. Phylogenetic analyses of Phytophthora endoglucanases of family 12 and other known members of EGL 12, revealed a close relatedness with a fairly conserved gene sub-family containing, among others, sequences from the fungi Emericella desertorum and Aspergillus aculeatus. This is the first report of family 12 EGLs present in plant pathogenic eukaryotes.     

Comparison of gene structures and enzymatic properties between two endoglucanases from Humicola grisea

We have cloned two endoglucanase genes (egl3 and egl4) from a thermophilic fungus, Humicola grisea. The coding region of the egl3 gene was interrupted by an intron of 56-bp, and the deduced amino acid sequence of the egl3 gene was 305 amino acids in length and showed 98.4% identity with Humicola insolens EGV. The coding region of the egl4 gene was also interrupted by an intron of 173-bp, which contains 34 TTC repeated sequence units, and the deduced amino acid sequence of the egl4 gene was 227 amino acids in length and showed 61.5% identity with H. grisea EGL3. The typical hinge and the cellulose-binding domain were observed in the C-terminal region of EGL3, but they were not observed in EGL4. In the 5′ upstream region of both genes, there were a TATA box or its similar sequence, CAAT motifs, and 6-bp sites which are identical or similar to the consensus sequence for binding a catabolite repressor CREA in Aspergillus nidulans. The egl3 and the egl4 genes were expressed in Aspergillus oryzae, and the translation products were purified. The fusion protein, EGL4CBD, which consists of a catalytic domain of EGL4 and the C-terminal region of EGL3, was also constructed and produced by A. oryzae, and purified. These enzymes showed relatively high activity toward carboxymethyl cellulose (CMC) and could not hydrolyze p-nitrophenyl-β-d-glucoside and p-nitrophenyl-β-d-cellobioside. The positive effect of substituting the C-terminal region of EGL4 with that of EGL3 was observed in the hydrolysis of CMC.     

Specific characteristics of family 45 endoglucanases from Mucorales in the use of textiles and laundry

We examined the characteristics of family 45 endoglucanases (glycoside hydrolases family 45; GH45) from Mucorales belonging to Zygomycota in the use of textiles and laundry. The defibrillation activities on lyocell fabric of family 45 endoglucanases from Mucorales, such as RCE1 and RCE2 from Rhizopus oryzae, MCE1 and MCE2 from Mucor circinelloides, and PCE1 from Phycomyces nitens, were much higher than those of the other family 45 endoglucanases. By contrast, family 45 endoglucanases from Mucorales were less resistant to anionic surfactant and oxidizing agent, main components in detergents, than the other family 45 endoglucanases. RCE1 consists of two distinct modules, a catalytic module and a carbohydrate-binding module family 1 (CBM1), and these common specific characteristics were considered to due to the catalytic module, but not to the CBM1.     

Nucleotide sequence of the celC gene encoding endoglucanase C of Clostridium thermocellum

The nucleotide sequence of the cellulase gene celC, encoding endoglucanase C of Clostridium thermocellum, has been determined. The coding region of 1032 bp was identified by comparison with the N-terminal amino acid (aa) sequence of endoglucanase C purified from Escherichia coli. The ATG start codon is preceded by an AGGAGG sequence typical of ribosome-binding sites in Gram-positive bacteria. The derived amino acid sequence corresponds to a protein of Mr 40,439. Amino acid analysis and apparent Mr of endoglucanase C are consistent with the amino acid sequence as derived from the DNA sequencing data. A proposed N-terminal 21-aa residue leader (signal) sequence differs from other prokaryotic signal peptides and is non-functional in E. coli. Most of the protein bears no resemblance to the endoglucanases A, B, and D of the same organism. However, a short region of homology between endoglucanases A and C was identified, which is similar to the established active sites of lysozymes and to related sequences of fungal cellulases.     

Isolation and analysis of two cellulase cDNAs from Orpinomyces joyonii

Two cellulase cDNAs, celB29 and celB2, were isolated from a cDNA library derived from mRNA extracted from the anaerobic fungus, Orpinomyces joyonii strain SG4. The nucleotide sequences of celB2 and celB29 and the primary structures of the proteins encoded by these cDNAs were determined. The larger celB29 cDNA was 1966bp long and encoded a 477 amino acid polypeptide with a molecular weight of 54kDa. Analysis of the 1451bp celB2 cDNA revealed an 1164bp open reading frame coding for a 44kDa protein consisting of 388 amino acids. Both deduced proteins had a high sequence similarity in central regions containing putative catalytic domains. Primary structure analysis revealed that CelB29 contained a Thr/Pro-rich sequence that separated the N-terminal catalytic domain from a C-terminal reiterated region of unknown function. Homology analysis showed that both enzymes belong to glycosyl hydrolase family 5 and were most closely related to endoglucanases from the anaerobic fungi Neocallimastic patriciarum, Neocallimastix frontalis and Orpinomyces sp. The classification of CelB29 and CelB2 as endoglucanases was supported by enzyme assays. The cloned enzymes had high activities towards barley beta-glucan, lichenan and carboxymethylcellulose (CMC), but not Avicel, laminarin, pachyman, xylan and pullulan. In addition, CelB29 and CelB2 showed activity against p-nitrophenyl-beta-D-cellobioside (pNP-G(2)) to p-nitrophenyl-beta-D-cellopentaoside (pNP-G(5)) but not p-nitrophenyl-beta-D-glucopyranoside (pNP-G(1)) with preferential activity against p-nitrophenyl-beta-D-cellotrioside (pNP-G(3)). Based on these results, we proposed that CelB29 and CelB2 are endoglucanases with broad substrate specificities for short- and long-chain beta-1,4-glucans.     

A single lysine in the N-terminal region of store-operated channels is critical for STIM1-mediated gating

Store-operated Ca(2+) entry is controlled by the interaction of stromal interaction molecules (STIMs) acting as endoplasmic reticulum ER Ca(2+) sensors with calcium release-activated calcium (CRAC) channels (CRACM1/2/3 or Orai1/2/3) in the plasma membrane. Here, we report structural requirements of STIM1-mediated activation of CRACM1 and CRACM3 using truncations, point mutations, and CRACM1/CRACM3 chimeras. In accordance with previous studies, truncating the N-terminal region of CRACM1 or CRACM3 revealed a 20-amino acid stretch close to the plasma membrane important for channel gating. Exchanging the N-terminal region of CRACM3 with that of CRACM1 (CRACM3-N(M1)) results in accelerated kinetics and enhanced current amplitudes. Conversely, transplanting the N-terminal region of CRACM3 into CRACM1 (CRACM1-N(M3)) leads to severely reduced store-operated currents. Highly conserved amino acids (K85 in CRACM1 and K60 in CRACM3) in the N-terminal region close to the first transmembrane domain are crucial for STIM1-dependent gating of CRAC channels. Single-point mutations of this residue (K85E and K60E) eliminate store-operated currents induced by inositol 1,4,5-trisphosphate and reduce store-independent gating by 2-aminoethoxydiphenyl borate. However, short fragments of these mutant channels are still able to communicate with the CRAC-activating domain of STIM1. Collectively, these findings identify a single amino acid in the N terminus of CRAC channels as a critical element for store-operated gating of CRAC channels.     

Nucleotide sequence of an endo-1,4-β-glucanase gene (celA) from Clostridium josui

The nucleotide sequence of a DNA fragment containing an endo-1,4-β-glucanase (EG-1) gene of Clostridium josui was determined by the dideoxy-chain termination method. The EG-1 coding sequence was an open reading frame encoding 369 amino acids, and a putative promoter sequence was located in the upstream region of the open reading frame. The N-terminal amino acid sequence of the endoglucanase (EG-1C) purified from the Escherichia coli transformant (JM103/pUCJ1) was consistent with the deduced sequence from ³⁰Val to ⁴⁴Lys. The estimated molecular weights of the precursor and the mature enzymes were 41,774 and 38,352, respectively. The region of amino acids from 61st to 335th of the enzyme revealed high homology with those of Bacillus sp. and Clostridium acetobutylicum endoglucanases.     

The N-terminal region of acyl-CoA synthetase 3 is essential for both the localization on lipid droplets and the function in fatty acid uptake

Cytosolic lipid droplets (LDs) are storage organelles for neutral lipids derived from endogenous metabolism. Acyl-CoA synthetase family proteins are essential enzymes in this biosynthetic pathway, contributing activated fatty acids. Fluorescence microscopy showed that ACSL3 is localized to the endoplasmic reticulum (ER) and LDs, with the distribution dependent on the cell type and the supply of fatty acids. The N-terminus of ACSL3 was necessary and sufficient for targeting reporter proteins correctly, as demonstrated by subcellular fractionation and confocal microscopy. The N-terminal region of ACSL3 was also found to be functionally required for the enzyme activity. Selective permeabilization and in silico analysis suggest that ACSL3 assumes a hairpin membrane topology, with the N-terminal hydrophobic amino acids forming an amphipathic helix restricted to the cytosolic leaflet of the ER membrane. ACSL3 was effectively translocated from the ER to nascent LDs when neutral lipid synthesis was stimulated by the external addition of fatty acids. Cellular fatty acid uptake was increased by overexpression and reduced by RNA interference of ACSL3. In conclusion, the structural organization of ACSL3 allows the fast and efficient movement from the ER to emerging LDs. ACSL3 not only esterifies fatty acids with CoA but is also involved in the cellular uptake of fatty acids, presumably indirectly by metabolic trapping. The unique localization of the acyl-CoA synthetase ACSL3 on LDs suggests a function in the local synthesis of lipids.     

The primary structure of human gamma-glutamyl transpeptidase

A cDNA hybridizable to that of rat gamma-glutamyl transpeptidase (GGT) was cloned from a cDNA library of human fetal liver. The insert of the cDNA clone contained 1866 bp consisting of an open reading frame (ORF) of 1709 bp (569 amino acids (aa), N-terminal portion truncated) and a 135-bp 3'-untranslated region followed by a polyadenylated tail. In parallel, amino acid sequences of N-terminal portions of heavy and light chains of a purified human GGT were determined. Two stretches of amino acid sequences identical to the N-terminal sequences of heavy and light chains were found in the ORF. We therefore concluded that the clone is a cDNA for human GGT. From the amino acid sequence deduced from cDNA, the heavy and the light chains of the purified enzyme are estimated to be composed of 351 aa (Mr 38,336) and of 189 aa (Mr 20,000), respectively. The heavy chain is preceded by a signal peptide of at least 29 aa presumed to be cleaved by bromelain treatment. Six putative N-glycosylation sites are present in the heavy subunit region and one in the light subunit region. Primary structure and hydrophobicity profile are closely similar to those of rat GGT.     

A homologue of EGL1 encoding endo-1,4-beta-glucanase in elongating pea stems

A gene (EGL2) encoding an endo-1,4-beta-glucanase in peas has been cloned as a homologue of EGL1. EGL2 encodes a polypeptide of 506 amino acids, including a 24-mer putative signal polypeptide. The gene product contains a domain conserved in endo-1,4-beta-glucanase (family 9) showing 60% amino acid identity to EGL1. EGL2 mRNA was accumulated only in the elongating regions of pea stems, although EGL1 mRNA was abundant in both elongating and non-elongating tissues. However, the level of EGL2 mRNA was not increased by the treatment with sucrose and auxin in pea segments. These results suggest that the expression of EGL2 either requires the presence of other factors related to the auxin effect or occurs independent of auxin in the elongating pea stems.     

The transmembrane segment of ryanodine receptor contains an intracellular membrane retention signal for Ca(2+) release channel.

The ryanodine receptor (RyR) is a large homotetrameric protein with a hydrophobic domain at the C-terminal end that resides in the endoplasmic reticulum (ER) or sarcoplasmic reticulum membrane and forms the conduction pore of a Ca(2+) release channel. Our previous studies showed that RyR expressed in heterologous cells localized to the ER membrane. Confocal microscopic imaging indicated that the ER retention signal is likely present within the C-terminal portion of RyR, a region that contains four putative transmembrane segments. To identify the amino acid sequence responsible for ER retention of RyR, we expressed fusion proteins containing intercellular adhesion molecule (ICAM), various fragments of RyR, and green fluorescent protein (GFP) in Chinese hamster ovary and COS-7 cells. ICAM is a plasma membrane-resident glycoprotein and serves as a reporter for protein trafficking to the cell surface membrane. Imaging analyses indicated that ICAM-GFP fusion proteins with RyR sequence preceding the four transmembrane segments, ICAM-RyR-(3661-3993)-GFP, and with RyR sequence corresponding to transmembrane segments 1, 2, and 3, ICAM-RyR-(4558-4671)-GFP and ICAM-RyR-(4830-4919)-GFP, were localized to the plasma membrane; fusion proteins containing the fourth transmembrane segment of RyR, ICAM-RyR-(4913-4943)-GFP, were retained in the ER. Biochemical assay showed that ICAM-RyR-GFP fusion proteins that target to the plasma membrane are fully glycosylated, and those retained in the intracellular membrane are core-glycosylated. Together our data indicate that amino acids 4918-4943 of RyR contain the signal sequence for ER retention of the Ca(2+) release channel.     

N-terminal portion of motilin determines its biological activity.

This study aimed to identify the portion of the 22 amino acid sequence of motilin responsible for the biological activity of the peptide. The contraction of rabbit duodenal muscle in vitro was measured when exposed to synthetic fragments of motilin corresponding to various sequences of the C- or N-terminal portions of the molecule. Fragments 2-22 or 3-22 (where the initial amino acids of the N-terminal ending were removed) were more than 1000 times less potent than the native molecule 1-22. Fragment 1-9 (where the last 13 amino acids located at the C-terminal side of motilin were removed) was devoid of any contractile capacity, while synthetic fragments whose C-terminal structure extended beyond the 1-9 motilin sequence maintained almost complete biological activity. N-terminal amino acid sequence 1-9 is therefore an essential determinant of the contractile activity of motilin.     

Assembly and routing of von Willebrand factor variants: the requirements for disulfide-linked dimerization reside within the carboxy-terminal 151 amino acids

The precursor protein of von Willebrand factor (pro-vWF) consists of four different repeated domains, denoted D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2, followed by a carboxy-terminal region of 151 amino acids without obvious internal homology. Previously, we have shown the requirement of the domains D1, D2, D', and D3 of pro-vWF in the assembly of pro-vWF dimers into multimers. Here, we define the domains of vWF involved in dimerization, using deletion mutants of full-length vWF cDNA transiently expressed in monkey kidney COS-1 cells. It is shown that only the carboxy-terminal 151 amino acid residues of vWF are required for dimerization. In addition, by analyzing a construct, encoding only the carboxy-terminal 151 amino acids of vWF, we find that the formation of dimers is an event independent of other domains present on pro-vWF, such as the domains C1 and C2 previously suggested to be involved in dimerization. Furthermore, it is shown that a deletion mutant of vWF, lacking the carboxy-terminal 151 amino acid residues and thus unable to dimerize, is proteolytically degraded in the ER. In contrast, a mutant protein, composed only of the carboxy-terminal 151 amino acids of vWF, and able to dimerize, is transported from the ER in a similar fashion as wild-type vWF. The role of the ER in the assembly of vWF is discussed with regard to the data presented in this paper on the intracellular fate of several vWF mutant proteins.     

Identification of an antimicrobial peptide from human methionine sulfoxide reductase B3

Human methionine sulfoxide reductase B3A (hMsrB3A) is an endoplasmic reticulum (ER) reductase that catalyzes the stereospecific reduction of methionine-R-sulfoxide to methionine in proteins. In this work, we identified an antimicrobial peptide from hMsrB3A protein. The N-terminal ER-targeting signal peptide (amino acids 1-31) conferred an antimicrobial effect in Escherichia coli cells. Sequence and structural analyses showed that the overall positively charged ER signal peptide had an Argand Pro-rich region and a potential hydrophobic α-helical segment that contains 4 cysteine residues. The potential α-helical region was essential for the antimicrobial activity within E. coli cells. A synthetic peptide, comprised of 2-26 amino acids of the signal peptide, was effective at killing Gram-negative E. coli, Klebsiella pneumoniae, and Salmonella paratyphi, but had no bactericidal activity against Gram-positive Staphylococcus aureus.