Characterization of an N-terminal secreted domain of the type-1 human metabotropic glutamate receptor produced by a mammalian cell line

A Chinese hamster ovary cell line has been established which secretes the N-terminal domain of human mGlu1 receptor. The secreted protein has been modified to contain a C-terminal hexa-histidine tag and can be purified by metal-chelate chromatography to yield a protein with an apparent molecular weight of 130 kDa. Following treatment with dithiothreitol the apparent molecular weight is reduced to 75 kDa showing that the protein is a disulphide-bonded dimer. N-terminal protein sequencing of both the reduced and unreduced forms of the protein yielded identical sequences, confirming that they were derived from the same protein, and identifying the site of signal-peptide cleavage of the receptor as residue 32 in the predicted amino acid sequence. Endoglycosidase treatment of the secreted and intracellular forms of the protein showed that the latter was present as an endoglycosidase H-sensitive dimer, indicating that dimerization is taking place in the endoplasmic reticulum. Characterization of the binding of [3H]quisqualic acid showed that the protein was secreted at levels of up to 2.4 pmol/mL and the secreted protein has a Kd of 5.6 +/- 1.8 nm compared with 10 +/- 1 nm for baby hamster kidney (BHK)-mGlu1alpha receptor-expressing cell membranes. The secreted protein maintained a pharmacological profile similar to that of the native receptor and the binding of glutamate and quisqualate were unaffected by changes in Ca2+ concentration.     

Isolation, characterization, and N-terminal sequence studies of cuticular proteins from the migratory locust, Locusta migratoria

The cuticle of the migratory locust, Locusta migratoria, contains more than a hundred different structural proteins, which can be extracted before but not after the cuticle is sclerotized. Fourteen of the proteins have been purified, covering a pI range of 6.4-10.6 and a molecular mass range of 15.2-36.8 kDa. The amino acid sequence from the N-terminal, ranging in length over 10-59 residues, have been obtained for eight of the proteins. A number of similarities, both in amino acid composition and in sequences, indicate that the proteins belong to a new protein family, characterized by an N-terminal part which is rich either in glycine, tyrosine and leucine or in hydrophilic amino acids, followed by a very alanine-rich portion. Similarities between this family of proteins and other structural proteins from insects are discussed.     

Isolation, Characterization, and Transcriptional Analysis of the Chitinase chi2 Gene (DQ011663) from the Biocontrol Fungus Metarhizium anisopliae var. anisopliae

Metarhizium anisopliae infects arthropods via a combination of specialized structures and cuticle degradation. Hydrolytic enzymes are accepted as key factors for the host penetration step and include chitinases. The characterization of the chi2 chitinase gene from M. anisopliae var. anisopliae is reported. The chi2 gene is interrupted by two short introns and is 1,542-bp long, coding a predicted protein of 419 amino acids with a stretch of 19 amino acid residues displaying characteristics of signal peptide. The predicted chitinase molecular mass is 44 kDa with a mature protein of 42 kDa and a theoretical pI of 4.8. The comparison of the CHI2 predicted protein to fungal orthologues revealed similarity to the glycohydrolase family 18 and a phylogenetic analysis was conducted. The chi2 gene is up-regulated by chitin as a carbon source and in conditions of fungus autolysis, and is down-regulated by glucose. This regulation is consistent with the presence of putative CreA/Crel/Crr1 carbon catabolic repressor binding domains on the regulatory sequence.     

Purification and characterization of two isozymes of polygalacturonase from Botrytis cinerea. Effect of calcium ions on polygalacturonase activity

The phytopathogenic fungus Botrytis cinerea produces a set of polygalacturonases (PGs) which are involved in the enzymatic degradation of pectin during plant tissue infection. Two polygalacturonases secreted by B. cinerea in seven-day-old liquid culture were purified to apparent homogeneity by chromatography. PG I was an exopolygalacturonase of molecular weight 65 kDa and pI 8.0 and PG II was an endopolygalacturonase of 52 kDa and pI 7.8. Enzymatic activity of PG I and PG II was partially inhibited by 1 mM CaCl2, probably by calcium chelation of polygalacturonic acid, the substrate of the enzyme.     

An isozyme of the NADP-malic enzyme of a CAM plant, Aloe arborescens, with variation on conservative amino acid residues

In Aloe arborescens, an obligate CAM plant, Western analysis detected three major isoforms of NADP-malic enzyme (NADP-ME), 72kDa with a pI of 6.0, 65kDa with a pI of 5.6 and 65kDa with a pI of 5.5. Among them, the 65kDa protein with a pI of 5.5 was leaf-specific, and the 65kDa protein with a pI of 5.6 was found only in roots, whereas the 72kDa protein was uniformly detected in both organs. Activity staining indicated enzyme activity of both 65kDa NADP-MEs but little activity of the 72kDa protein. A cDNA clone encoding a leaf-abundant NADP-ME, AME1, was isolated. Deduced amino acid sequence of AME1 showed a high degree of homology to known NADP-MEs, but it was also found that AME1 contained substitutions on five conservative amino acid residues, some of which have been predicted to be important for their enzyme activity. Transgenic rice carrying the aloe AME1 gene efficiently produced an additional 65kDa protein with a pI of 5.5 as an active NADP-ME. These results indicate that AME1 corresponds to the leaf-specific 65kDa NADP-ME, which may be involved in CAM photosynthesis. It was also shown that substitutions of these conservative amino acid residues identified in AME1 still allowed it to give enzyme activity.     

Purification and characterization of a glucoamylase secreted by the plant pathogen Scierotinia sclerotiorum

Among the lytic enzymes secreted by the phytopathogen fungus Sclerotinia sclerotiorum, a starch-degrading enzyme has been isolated and characterized. This glycoprotein of 72 kDa is composed of several isoforms ranging from pI 4.8 to 5.4. The enzymatic parameters have been determined. Specificity studies together with the analysis of the reaction products show that it is an alpha-1,4-glucanohydrolase. This result is also corroborated by the analysis of the N-terminal and two inner amino acids sequences that are very similar to fungal glucoamylase genes or enzymes so far sequenced.     

Isolation of a 43 kDa protein from Mycobacterium tuberculosis H37Rv and its identification as a pyridine nucleotide transhydrogenase

R.G. DESHPANDE, M.B. KHAN, D.A. BHAT AND R.G. NAVALKAR. 1994. A 43 kDa protein (TB43) was isolated from the cell sonicate (CS) of Mycobacterium tuberculosis H₃₇Rv with immobilized metal affinity chromatography (IMAC) on a Ni-nitrilotriacetic acid column. Two-dimensional electrophoresis of the IMAC fraction showed a major spot with an M_r of 43000 and a pI of ˜6.0. The N-terminal amino acid sequence of TB43 was met-arg-val-gly-ile-pro-asn-glu-thr-lys-asn-asn-glu-phe-arg-val-ala-ile-thr-pro-ala. It showed 86% homology with the N-terminal end of the alanine dehydrogenase of Myco. tuberculosis and 65% homology with the N-terminal end of the alpha-subunit of the Escherichia coli pyridine nucleotide transhydrogenase (Tsh). TB43 did not show any alanine dehydrogenase activity and did not react with monoclonal antibody (MAb) HBT10, which is known to recognize the 40 kDa alanine dehydrogenase of Myco. tuberculosis. It was also not recognized by MAb F29–29 which is known to react with a 43 kDa protein of Myco. tuberculosis complex. This protein exhibited strong Tsh activity. A similar 43 kDa protein showing Tsh activity was also isolated by IMAC from Myco. bovis CS. However, the pI of the protein was ˜7.0. A similar protein could not be isolated from the CS or culture filtrate of Myco. bovis BCG and Myco. tuberculosis H₃₇Ra. TB43 is a cell-associated pyridine nucleotide transhydrogenase and is distinct from the 40/44 kDa secreted alanine dehydrogenase of Myco. tuberculosis.     

A novel thermostable branching enzyme from an extremely thermophilic bacterial species, Rhodothermus obamensis

A branching enzyme (EC 2.4.1.18) gene was isolated from an extremely thermophilic bacterium, Rhodothermus obamensis. The predicted protein encodes a polypeptide of 621 amino acids with a predicted molecular mass of 72 kDa. The deduced amino acid sequence shares 42-50% similarity to known bacterial branching enzyme sequences. Similar to the Bacillus branching enzymes, the predicted protein has a shorter N-terminal amino acid extension than that of the Escherichia coli branching enzyme. The deduced amino acid sequence does not appear to contain a signal sequence, suggesting that it is an intracellular enzyme. The R. obamensis branching enzyme was successfully expressed both in E. coli and a filamentous fungus, Aspergillus oryzae. The enzyme showed optimum catalytic activity at pH 6.0-6.5 and 65 degrees C. The enzyme was stable after 30 min at 80 degrees C and retained 50% of activity at 80 degrees C after 16 h. Branching activity of the enzyme was higher toward amylose than toward amylopectin. This is the first thermostable branching enzyme isolated from an extreme thermophile.     

Purification and first characterization of the secreted and cellular 52-kDa proteins regulated by estrogens in human-breast cancer cells

An estrogen-regulated 52-kDa glycoprotein secreted by MCF7 breast cancer cells was first purified from serum-free conditioned medium by concanavalin-A--Sepharose (ConA--Sepharose). The 13% pure protein was then used to obtain monoclonal antibodies to the 52-kDa protein [Garcia et al. (1985) Cancer Res. 45, 709-716]. Using ConA--Sepharose and monoclonal antibody affinity chromatographies, the secreted 52-kDa protein was finally purified to homogeneity as verified by silver staining of sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and one single N-terminal amino acid. The purification factor was approximately 1400 and the yield 40%. The same two-step procedure, applied to MCF7 cell extracts, yielded four immunologically related proteins of 52 kDa, 48 kDa, 34 kDa and 17 kDa, which were purified 1250-fold with a yield of 30%. These components were further separated by high-performance liquid chromatography gel filtration under denaturing conditions. The final products were homogeneous on the basis of silver-stained SDS-PAGE and gel filtration. However, isoelectrofocusing showed that the pI of the secreted 52-kDa protein and the cellular 34-kDa protein varied from 5.5 to 6.5. Amino acid analysis of the secreted and the related cellular 34-kDa protein is given. Western immunoblotting, pulse chase studies and post-translational studies indicate that the 52-kDa protein is the precursors of a lysosomal enzyme which is partially secreted and partially processed into smaller cellular forms.     

Structural determinants of processing and secretion of the Haemophilus influenzae Hap protein

Haemophilus influenzae elaborates a surface protein called Hap, which is associated with the capacity for intimate interaction with cultured epithelial cells. Expression of hap results in the production of three protein species: outer membrane proteins of approximately 155 kDa and 45 kDa and an extracellular protein of approximately 110 kDa. The 155 kDa protein corresponds to full-length mature Hap (without the signal sequence), and the 110 kDa extracellular protein represents the N-terminal portion of mature Hap (designated Hap_s). In the present study, we examined the mechanism of processing and secretion of Hap. Site-directed mutagenesis suggested that Hap is a serine protease that undergoes autoproteolytic cleavage to generate the 110 kDa extracellular protein and the 45 kDa outer membrane protein. Biochemical analysis confirmed this conclusion and established that cleavage occurs on the bacterial cell surface. Determination of N-terminal amino acid sequence and mutagenesis studies revealed that the 45 kDa protein corresponds to the C-terminal portion of Hap, starting at N1037. Analysis of the secondary structure of this protein (designated Hap_β) predicted formation of a β-barrel with an N-terminal transmembrane α-helix followed by 14 transmembrane β-strands. Additional analysis revealed that the final β-strand contains an amino acid motif common to other β-barrel outer membrane proteins. Upon deletion of this entire C-terminal consensus motif, Hap could no longer be detected in the outer membrane, and secretion of Hap_s was abolished. Deletion or complete alteration of the final three amino acid residues had a similar but less dramatic effect, suggesting that this terminal tripeptide is particularly important for outer membrane localization and/or stability of the protein. In contrast, isolated point mutations that disrupted the amphipathic nature of the consensus motif or eliminated the C-terminal tryptophan had no effect on outer membrane localization of Hap or secretion of Hap_s. These results provide insight into a growing family of Gram-negative bacterial exoproteins that are secreted by an IgA1 protease-like mechanism; in addition, they contribute to a better understanding of the structural determinants of targeting of β-barrel proteins to the bacterial outer membrane.     

A novel proteinase inhibitor gene transiently induced by tobacco mosaic virus infection

A gene (NgPI) encoding a novel proteinase inhibitor (PI) has been isolated from tobacco leaves. Protein encoded by the gene consists of 241 amino acid residues having a predicted molecular mass of 26.7 kDa and a calculated pI of 8.7. A predicted N-terminal signal sequence followed by a vacuolar targeting signal and a peptide conserved in the Kunitz type PIs were identified. The deduced NgPI protein has sequence homology with aspartic and cysteine protease inhibitors. The gene is present as double copies in the Nicotiana glutinosa genome. Expression of the NgPI gene is rapidly and transiently induced by tobacco mosaic virus infection at a time earlier than apparent lesions of hypersensitive responses appear on the leaves.     

Manganese-containing superoxide dismutase and its gene from Candida albicans

Mitochondrial manganese-containing superoxide dismutase was purified around 112-fold with an overall yield of 1.1% to apparent electrophoretic homogeneity from the dimorphic pathogenic fungus, Candida albicans. The molecular mass of the native enzyme was 106 kDa and the enzyme was composed of four identical subunits with a molecular mass of 26 kDa. The enzyme was not sensitive to either cyanide or hydrogen peroxide. The N-terminal amino acid sequence alignments (up to the 18th residue) showed that the enzyme has high similarity to the other eukaryotic manganese-containing superoxide dismutases. The gene sod2 encoding manganese-containing superoxide dismutase has been cloned using a product obtained from polymerase chain reaction. Sequence analysis of the sod2 predicted a manganese-containing superoxide dismutase that contains 234 amino acid residues with a molecular mass of 26173 Da, and displayed 57% sequence identity to the homologue of Saccharomyces cerevisiae. The deduced N-terminal 34 amino acid residues may serve as a signal peptide for mitochondrial translocation. Several regulatory elements such as stress responsive element and haem activator protein 2/3/4/5 complex binding sites were identified in the promoter region of sod2. Northern analysis with a probe derived from the cloned sod2 revealed a 0.94-kb band, which corresponds approximately to the expected size of mRNA deduced from sod2.     

Cloning, sequence analysis and expression of Pseudoalteromonas elyakovii IAM 14594 gene (alyPEEC) encoding the extracellular alginate lyase.

A gene (alyPEEC) encoding an alginate lyase of Pseudoalteromonas elyakovii IAM 14594 was cloned using the plasmid vector pUC118 and expressed in Escherichia coli. Sequencing of a 3.0kb fragment revealed a 1,197bp open reading frame encoding 398 amino acid residues. The calculated molecular mass and isoelectric point of the alyPEEC gene product are 43.2 kDa and pI 5.29. A region G(165) to V(194) in the AlyPEEC internal sequence is identical to the N-terminal amino acid sequence of the previously purified extracellular alginate lyase of P. elyakovii, and the calculated molecular mass (25.4 kDa) and isoelectric point (pI 4.78) of the region resembled those of the purified enzyme. Expression of enzymically-active alginate lyase from alyPEEC required growth of recombinant E. coli in LB broth containing 50% (v/v) artificial seawater (ASW). Alginate lyase activity with broad substrate specificity was detected in both 42 and 30 kDa products. Subcloning of the region G(165) to N(398) of AlyPEEC corresponding to the 30 kDa protein confirmed that this region of the alyPEEC gene encoded the active site of the enzyme. A region A(32) to G(164) corresponding to about 13 kDa of the N-terminal region of AlyPEEC showed about 30% identity to a putative chitin binding domain of Streptomyces chitinases, but did not exhibit any catalytic activity.     

Purification and characterization of two isozymes of polygalacturonase from Botrytis cinerea. Effect of calcium ions on polygalacturonase activity

The phytopathogenic fungus Botrytis cinerea produces a set of polygalacturonases (PGs) which are involved in the enzymatic degradation of pectin during plant tissue infection. Two polygalacturonases secreted by B. cinerea in seven-day-old liquid culture were purified to apparent homogeneity by chromatography. PG I was an exopolygalacturonase of molecular weight 65 kDa and pI 8.0 and PG II was an endopolygalacturonase of 52 kDa and pI 7.8. Enzymatic activity of PG I and PG II was partially inhibited by 1 mM CaCl₂, probably by calcium chelation of polygalacturonic acid, the substrate of the enzyme.     

Complementary DNA sequence of rabbit CAP18--a unique lipopolysaccharide binding protein

CAP18 is a novel 18 kDa cationic protein [pI approximately 10] originally purified from rabbit granulocytes using as an assay the agglutination of lipopolysaccharide (LPS) coated erythrocytes. cDNA clones encoding CAP18 were isolated from a rabbit bone marrow cDNA library using a PCR generated oligonucleotide probe derived from the N-terminal amino acid sequence. The deduced amino acid sequence reveals a putative signal sequence of 29 amino acids and a mature protein of 142 amino acid residues. The predicted size of the encoded protein is 16.6 kDa with a pI of 10. There are no N-linked glycosylation sites. The CAP18 sequence bears no homology with other known LPS-binding proteins including human bacterial permeability increasing protein (BPI)(1) and rabbit LPS binding protein (LBP)(2).     

Purification,characterization and molecular cloning of Vibrio fluvialis hemolysin

Hemolysin of Vibrio fluvialis (VFH) was purified from culture supernatants by ammonium sulfate precipitation and successive column chromatographies on DEAE-cellulose and Mono-Q. N-terminal amino acid sequences of the purified VFH were determined. The purified protein exhibited hemolytic activity on many mammalian erythrocytes with rabbit erythrocytes being the most sensitive to VFH. Activity of the native VFH was inhibited by the addition of Zn2+, Ni2+, Cd2+ and Cu2+ ions at low concentrations. Pores formed on rabbit erythrocytes were approximately 2.8-3.7 nm in diameter, as demonstrated by osmotic protection assay. Nucleotide sequence analysis of the vfh gene revealed an open reading frame (ORF) consisting of 2200 bp which encodes a protein of 740 amino acids with a molecular weight of 82 kDa. Molecular weight of the purified VFH was estimated to be 79 kDa by SDS-PAGE and N-terminal amino acid sequence revealed that the 82 kDa prehemolysin is synthesized in the cytoplasm and is then secreted into the extracellular environment as the 79 kDa mature hemolysin after cleavage of 25 N-terminal amino acids. Deletion of 70 amino acids from the C-terminus exhibited a smaller hemolytic activity, while deletion of 148 C-terminal amino acids prevented hemolytic activity.