A new type of mitogenic factor produced by Streptococcus pyogenes.

A new type of mitogenic factor (protein) was purified from the culture supernatant of a strain of Streptococcus pyogenes by SP-Sephadex C-25 column chromatography, preparative isoelectric focusing and reversed-phase high-performance liquid chromatography. The purified factor, showing marked mitogenic activity in rabbit peripheral blood lymphocytes, gave a single-band staining for protein on SDS-PAGE. The molecular weight of the purified mitogenic factor was determined to be 25,370, which was different from those calculated from reported amino acid sequences deduced from 4 different nucleotide sequences of 3 kinds of streptococcal pyrogenic exotoxins (two SPEAs, SPEB and SPEC). The amino acid sequence of the N-terminal region of the purified mitogenic factor was determined to be Gln-Thr-Gln-Val-Ser-Asn-Asp-Val-Val-Leu-Asn-Asp-Gly-Ala-Ser-Lys-Tyr-Leu- Asn-Glu - Ala-, which was also different from the reported N-terminal sequences deduced from the 4 different nucleotide sequences. These data indicate that this mitogenic factor is distinct from the already described streptococcal pyrogenic exotoxins.     

Use of flow cytometry for the adhesion analysis of Streptococcus pyogenes mutant strains to epithelial cells: investigation of the possible role of surface pullulanase and cysteine protease, and the transcriptional regulator Rgg

Background  

Flow cytometry based adherence assay is a potentially powerful but little used method in the study of bacterial binding to host structures. We have previously characterized a glycoprotein-binding activity in Streptococcus pyogenes called 'strepadhesin' binding to thyroglobulin, submaxillar mucin, fetuin and asialofetuin. We have identified surface-associated pullulanase (PulA) and cysteine protease (SpeB) as carriers of strepadhesin activity. In the present paper, we investigated the use of flow cytometry as a method to study the binding of Rgg, SpeB and PulA knock-out strains to cultured human epithelial cells.     

Hormonal regulation, processing, and secretion of cysteine proteinases in barley aleurone layers.

Barley aleurone layers synthesize and secrete several proteases in response to gibberellic acid (GA3). Two major cysteine proteinases designated EP-A (37,000 M(r)) and EP-B (30,000 M(r)) have been described [Koehler and Ho (1988). Plant Physiol. 87, 95-103]. We now report the cDNA cloning of EP-B and describe the post-translational processing and hormonal regulation of both cysteine proteinases. Three cDNAs for cysteine proteinases were cloned from GA3-induced barley aleurone layers. Genomic DNA gel blot analysis indicated that these are members of a small gene family with no more than four to five different genes. The proteins encoded by two of these clones, pHVEP1 and 4, are 98% similar to each other and are isozymes of EP-B. The proteins contain large preprosequences followed by the amino acid sequence described as the mature N terminus of purified EP-B, and are antigenic to EP-B antiserum. The results of pulse-chase experiments indicated that the post-translational processing of large prosequences proceeds in a multistep fashion to produce the mature enzymes. Processing intermediates for EP-B are observed both in the aleurone layers and surrounding incubation medium, but only mature EP-A is secreted. The regulation of synthesis of EP-A, EP-B, and other aleurone cysteine proteinases was compared at the protein and mRNA levels. We conclude that barley aleurone cysteine proteinases are differentially regulated with respect to their temporal and hormonally induced expression.     

Site-directed mutation of conserved cysteine residues does not inactivate the Streptococcus pyogenes hyaluronan synthase.

Hyaluronan synthase (HAS), the enzyme responsible for the production of hyaluronic acid (HA), is a well-conserved membrane-bound protein in both prokaryotes and eukaryotes. This enzyme performs at least six discrete functions in producing a heterodisaccharide polymer of several million molecular weight and extruding it from the cell. Among the conserved motifs and domains within the Class I HAS family are four cysteine residues. Cysteines in many proteins are important in establishing and maintaining tertiary structure or in the coordination of catalytic functions. In the present study we utilized a combination of site-directed mutagenesis, chemical labeling, and kinetic analyses to determine the importance of specific Cys residues for catalysis and structure of the HA synthase from Streptococcus pyogenes (spHAS). The enzyme activity of spHAS was partially inhibited by cysteine-reactive chemical reagents such as N-ethylmaleimide. Quantitation of the number of Cys residues modified by these reagents, using MALDI-TOF mass spectrometry, demonstrated that there are no stable disulfide bonds in spHAS. The six Cys residues of spHAS were then mutated, individually and in various combinations, to serine or alanine. The single Cys-mutants were all kinetically similar to the wild-type enzyme in terms of their V(max) and K(m) values for HA synthesis. The Cys-null mutant, in which all Cys residues were mutated to alanine, retained approximately 66% of wild-type activity, demonstrating that despite their high degree of conservation within the HAS family, Cys residues are not absolutely necessary for HA biosynthesis by the spHAS enzyme.     

HrcA is a negative regulator of the dnaK and groESL operons of Streptococcus pyogenes

The genome of Streptococcus pyogenes, an important human pathogen, encodes homologs of the principal bacterial heat shock proteins DnaK and GroES, -EL, as well as HrcA, a negative regulator of dnaK and groESL expression in other Gram-positive bacteria. Using nuclease protection assays to measure dnaK/groESL mRNA abundance and a non-polar insertion to disrupt hrcA, we demonstrate that heat shock triggers a 4- to 8-fold increase in dnaK and groESL-specific mRNAs within 5 min of the temperature shift and that HrcA is a negative regulator of S. pyogenes dnaK/groESL mRNA abundance in unstressed S. pyogenes. Although the loss of HrcA elevated dnaK and groESL mRNA levels under non-heat shock conditions, the relative abundance of these RNAs increased further in heat shocked S. pyogenes, suggesting an additional element contributing to their synthesis or stability.     

A bacterial factor induces changes in cysteine proteinase forms in the cellular slime mould Dictyostelium discoideum.

The electrophoretic pattern of cysteine proteinases in axenically grown myxamoebae of Dictyostelium discoideum can be altered by the addition of either Gram-negative (Klebsiella aerogenes, Escherichia coli) or Gram-positive (Micrococcus lysodeikticus, Bacillus subtilis) bacteria to the culture. No changes occurred, however, if either yeast or latex beads were used in place of bacteria. The changes involved the simultaneous loss of proteinases characteristic of the axenic cells (the A-forms) and the acquisition of those found in cells which have been grown on bacteria (the B-forms). Using K. aerogenes the conversion was complete within 4 h. Extracellular proteinase activity was unaffected during this period. After the D. discoideum cells had been lysed, no equivalent change in proteinase band pattern could be produced either by prolonged incubation of cell extracts or by treatment with proteinases. An identical conversion could be induced in cultures of myxamoebae by a factor, cysteine proteinase converting factor (CPCF), present in the 15,000 g supernatant of a sonicated suspension of K. aerogenes. CPCF was macromolecular, as demonstrated by both ultrafiltration and gel filtration, acid-precipitable, but was soluble in ethanol or alkali. Its activity was unaffected by treatment with trypsin. The results suggested that CPCF might be a component of the bacterial cell wall, and since its activity was affected by lysozyme treatment, peptidoglycan is implicated. The results can be interpreted in terms of a novel nutrient-dependent post-translational change which affected most of the cysteine proteinases present in D. discoideum myxamoebae.     

Affinity purification of the novel cysteine proteinase papaya proteinase IV, and papain from papaya latex.

A procedure is described for the purification of a previously undetected cysteine proteinase, which we have called papaya proteinase IV, from spray-dried latex of the papaya (Carica papaya) plant. The purification involves affinity chromatography on Gly-Phe-aminoacetonitrile linked to CH-Sepharose 4B, with elution by 2-hydroxyethyl disulphide at pH 4.5. The product thus obtained is a mixture of almost fully active papain and papay proteinase IV, which are then separated by cation-exchange chromatography. A preliminary characterization of papaya proteinase IV showed it to be very similar to chymopapain in both molecular size and charge. However, the new enzyme is immunologically distinct from the previously characterized cysteine proteinases of papaya latex. It also differs in its lack of activity against the synthetic substrates of the other papaya proteinases, in its narrow specificity against protein substrates and its lack of inhibition by chicken cystatin. Papaya proteinase IV is abundant, contributing almost 30% of the protein in spray-dried papaya latex, and contamination of chymopapain preparations with this enzyme may account for some of the previously reported heterogeneity of chymopapain.     

Deoxyribonuclease-sensitive transfer of an R plasmid in Streptococcus pyogenes (group A)

Abstract The 11.6-megadalton (MDa) plasmid pIP955, encoding resistance to macrolides and related drugs and chloramphenicol, harbored by Streptococcus pyogenes strain A449, transferred only by filter mating and only into Streptococcus sanguis strain Challis recipient. This transfer was completely inhibited by low concentrations of deoxyribonuclease I and occurred even if the donor strain had been killed.     

A role for ADP-ribosylation factor 6 in the processing of G-protein-coupled receptors

After agonist-induced internalization, the vasopressin V2 receptor (V2R) does not recycle to the plasma membrane. The ADP-ribosylation factor (ARF) proteins initiate vesicular intracellular traffic by promoting the recruitment of adaptor proteins; thus, we sought to determine whether ARF6 could promote V2R recycling. Neither the agonist-induced internalization nor the recycling of the V2R was regulated by ARF6, but a constitutively active mutant of ARF6 reduced cell-surface V2Rs 10-fold in the absence of agonist treatment. Visualization of the ARF6 mutant-expressing cells revealed a vacuolar-staining pattern of the V2R instead of the normal plasma membrane expression. Analysis of V2R maturation revealed that reduced cell-surface expression was due to the diminished ability of the newly synthesized receptor to migrate from the endoplasmic reticulum to the Golgi network. The same mechanism affected processing of the V1aR and acetylcholine M2 receptors. Therefore, ARF6 controls the exit of the V2 and other receptors from the endoplasmic reticulum in addition to its established role in the trafficking of plasma-membrane-derived vesicles.     

Purification, cloning and autoproteolytic processing of an aspartic proteinase from Centaurea calcitrapa.

Plant aspartic proteinases (APs) have been isolated from several seed and leaf sources but the only well characterized enzymes from flowers are cardosins and cyprosins from cardoon, Cynara cardunculus L. Here we report a full-length cDNA clone encoding an AP named cenprosin from the flowers of Centaurea calcitrapa L., a thistle related to cardoon. As found for all eukaryotic APs, the deduced primary sequence consists of a signal sequence, a propart and a mature enzyme. In addition, an internal sequence region of 104 residues typical only of plant APs (a plant-specific insert) is present in the primary structure. Northern analysis revealed that the strongest expression is in fresh flowers. The enzyme is also expressed in fairly high amounts in seeds and in leaves, a feature not detected for cardoon APs. The corresponding enzyme was purified in its precursor form from fresh flowers using ammonium-sulfate precipitation followed by ion-exchange and hydrophobic-interaction chromatography. The processing of the precursor into its mature form was studied in vitro. The enzyme underwent autocatalytic processing at pH 3.0 resulting in two chains of 16 and 30 kDa. When dried flowers were used as a starting material for purification, only 16- and 30-kDa chains were obtained, suggesting that autoproteolytic activation of procenprosin in vivo occurs mainly during drying of the flowers. This may indicate a specific degradative role for the enzyme during senescence of the flowers.     

The role of cysteine proteinase and carboxypeptidase in the breakdown of storage proteins in buckwheat seeds

Two proteolytic enzymes, a cysteine proteinase and a carboxypeptidase, responsible for breakdown of the main storage protein, 13S globulin, were purified from buckwheat seedlings (Fagopyrum esculentum Moench) by (NH₄)₂SO₄ fractionation, gel-filtration on Sephadex G-150, ionexchange chromatography on DEAE-Toyopearl 650 M and chromatofocusing. The cysteine proteinase was purified 74-fold. It has a pH optimum of 5.5, a pI of 4.5 and an apparent molecular mass (M_r) of 71000. The carboxypeptidase was purified 128-fold. It has a pH optimum of 5.3, a pI of 5.8 and a M_r of 78500. Cysteine proteinase hydrolyzed the modified 13S globulin only if the reaction products were eliminated from the incubation mixture by dialysis. Storage protein degradation by the proteinase increased in the presence of carboxypeptidase. We suggest that the two enzymes complete the digestion of 13S globulin after its preliminary hydrolysis by the earlier described enzyme, metalloproteinase, present in dry buckwheat seeds.Abbreviations BSA bovine serum albumin - DEAE diethylaminoethyl - M_r apparent molecular mass - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate