PubMed Health⌕ Search

Biomedical subjects

P L DeAngelis

Publications and source records attributed to P L DeAngelis.

At least 19 recordsLinked to original sources
PubMed2001

Functional molecular mass of a vertebrate hyaluronan synthase as determined by radiation inactivation analysis.

Hyaluronan (HA), a linear polysaccharide composed of N-acetylglucosamine-glucuronic acid repeats, is found in the extracellular matrix of vertebrate tissues as well as the capsule of several pathogenic bacteria. The HA synthases (HASs) are dual-action glycosyltransferases that catalyze the addition of two different sugars from UDP-linked precursors to the growing HA chain. The prototypical vertebrate hyaluronan synthase, xlHAS1 (or DG42) from Xenopus laevis, is a 588-residue membrane protein. Recently, the streptococcal enzyme was found to function as a monomer of protein with approximately 16 lipid molecules. The vertebrate enzymes are larger than the streptococcal enzymes; based on the vertebrate HAS deduced amino acid sequence, two additional membrane-associated regions at the carboxyl terminus are predicted. We have utilized radiation inactivation to measure the target size of yeast-derived recombinant xlHAS1. The target size of HAS activity was confirmed using two internal standards. First, samples were spiked with glucose-6-phosphate dehydrogenase, an enzyme of known molecular weight. Second, parallel samples of native xlHAS1 and a xlHAS1-green fluorescent protein fusion (833 residues) were compared; substantial confidence was gained by using this novel internal standard. Our test also corroborated the basic tenets of radiation inactivation theory. We found that the vertebrate HAS protein functions catalytically as a monomer.

Animals↗
PubMed2000

Topological organization of the hyaluronan synthase from Streptococcus pyogenes.

Since we first reported (DeAngelis, P. L., Papaconstantinou, J., and Weigel, P. H. (1993) J. Biol. Chem. 268, 19181-19184) the cloning of the hyaluronan (HA) synthase from Streptococcus pyogenes (spHAS), numerous membrane-bound HA synthases have been discovered in both prokaryotes and eukaryotes. The HASs are unique among enzymes studied to date because they mediate 6-7 discrete functions in order to assemble a polysaccharide containing hetero-disaccharide units and simultaneously effect translocation of the growing HA chain through the plasma membrane. To understand how the relatively small spHAS performs these various functions, we investigated the topological organization of the protein utilizing fusion analysis with two reporter enzymes, alkaline phosphatase and beta-galactosidase, as well as several other approaches. From these studies, we conclude that the NH2 terminus and the COOH terminus, as well as the major portion of a large central domain are localized intracellularly. The first two predicted membrane domains were confirmed to be transmembrane domains and give rise to a very small extracellular loop that is inaccessible to proteases. Several regions of the large internal central domain appear to be associated with, but do not traverse, the membrane. Following the central domain, there are two additional transmembrane domains connected by a second small extracellular loop that also is inaccessible to proteases. The COOH-terminal approximately 25% of spHAS also contains a membrane domain that does not traverse the membrane and may contain extensive re-entrant loops or amphipathic helices. Numerous membrane associations of this latter COOH-terminal region and the central domain may be required to create a pore-like structure through which a growing HA chain can be extruded to the cell exterior. Based on the high degree of similarity among Class I HAS family members, these enzymes may have a similar topological organization for their spHAS-related domains.

Base Sequence↗
PubMed2000

Identification and molecular cloning of a chondroitin synthase from Pasteurella multocida type F.

Pasteurella multocida Type F, the minor fowl cholera pathogen, produces an extracellular polysaccharide capsule that is a putative virulence factor. It was reported that the capsule was removed by treating microbes with chondroitin AC lyase. We found by acid hydrolysis that the polysaccharide contained galactosamine and glucuronic acid. We molecularly cloned a Type F polysaccharide synthase and characterized its enzymatic activity. The 965-residue enzyme, called P. multocida chondroitin synthase (pmCS), is 87% identical at the nucleotide and the amino acid level to the hyaluronan synthase, pmHAS, from P. multocida Type A. A recombinant Escherichia coli-derived truncated, soluble version of pmCS (residues 1-704) was shown to catalyze the repetitive addition of sugars from UDP-GalNAc and UDP-GlcUA to chondroitin oligosaccharide acceptors in vitro. Other structurally related sugar nucleotide precursors did not substitute in the elongation reaction. Polymer molecules composed of approximately 10(3) sugar residues were produced, as measured by gel filtration chromatography. The polysaccharide synthesized in vitro was sensitive to the action of chondroitin AC lyase but resistant to the action of hyaluronan lyase. This is the first report identifying a glycosyltransferase that forms a polysaccharide composed of chondroitin disaccharide repeats, [beta(1,4)GlcUA-beta(1,3)GalNAc](n). In analogy to known hyaluronan synthases, a single polypeptide species, pmCS, possesses both transferase activities.

Amino Acid Sequence↗
PubMed2000

Dissection of the two transferase activities of the Pasteurella multocida hyaluronan synthase: two active sites exist in one polypeptide.

Type A Pasteurella multocida, an animal pathogen, employs a hyaluronan [HA] capsule to avoid host defenses. PmHAS, the 972-residue membrane-associated hyaluronan synthase, catalyzes the transfer of both GlcNAc and GlcUA to form the HA polymer. To define the catalytic and membrane-associated domains, pmHAS mutants were analyzed. PmHAS1-703 is a soluble, active HA synthase suggesting that the carboxyl-terminus is involved in membrane association of the native enzyme. PmHAS1-650 is inactive as a HA synthase, but retains GlcNAc-transferase activity. Within the pmHAS sequence, there is a duplicated domain containing a short motif, Asp-Gly-Ser, that is conserved among many beta-glycosyltransferases. Changing this aspartate in either domain to asparagine, glutamate, or lysine reduced the HA synthase activity to low levels. The mutants substituted at residue 196 possessed GlcUA-transferase activity while those substituted at residue 477 possessed GlcNAc-transferase activity. The Michaelis constants of the functional transferase activity of the various mutants, a measure of the apparent affinity of the enzymes for the precursors, were similar to wild-type values. Furthermore, mixing D196N and D477K mutant proteins in the same reaction allowed HA polymerization at levels similar to the wild-type enzyme. These results provide the first direct evidence that the synthase polypeptide utilizes two separate glycosyltransferase sites.

Amino Acid Motifs↗
PubMed1999

Hyaluronan synthases: fascinating glycosyltransferases from vertebrates, bacterial pathogens, and algal viruses.

Hyaluronan (or hyaluronic acid or hyaluronate; HA) is a polysaccharide found in the extracellular matrix of vertebrate tissues and in the surface coating of certain Streptococcus and Pasteurella bacterial pathogens. At least one algal virus directs its host to produce HA on the cell surface early in infection. HA synthases (HASs) are the enzymes that polymerize HA using uridine diphospho-sugar precursors. In all known cases, HA is secreted out of the cell; therefore, HASs are normally found in the outer membranes of the organism. In the last 6 years, the HASs have been molecularly cloned from all the above sources. They were the first class of glycosyltransferases identified in which a single polypeptide species catalyzes the transfer of two different monosaccharides; this finding is in contrast to the usual 'single enzyme, single sugar' dogma of glycobiology. There appear to be two distinct classes of HASs based on differences in amino acid sequence, topology in the membrane, and reaction mechanism. This review discusses the current state of knowledge surrounding the molecular details of HA biosynthesis and summarizes the possible evolutionary history of the HASs.

Amino Acid Sequence↗
PubMed1999

Molecular directionality of polysaccharide polymerization by the Pasteurella multocida hyaluronan synthase.

Hyaluronan (HA), a long linear polymer composed of alternating glucuronic acid and N-acetylglucosamine residues, is an essential polysaccharide in vertebrates and a putative virulence factor in certain microbes. All known HA synthases utilize UDP-sugar precursors. Previous reports describing the HA synthase enzymes from Streptococcus bacteria and mammals, however, did not agree on the molecular directionality of polymer elongation. We show here that a HA synthase, PmHAS, from Gram-negative P. multocida bacteria polymerizes the HA chain by the addition of sugar units to the nonreducing terminus. Recombinant PmHAS will elongate exogenous HA oligosaccharide acceptors to form long polymers in vitro; thus far no other HA synthase has displayed this capability. The directionality of synthesis was established definitively by testing the ability of PmHAS to elongate defined oligosaccharide derivatives. Analysis of the initial stages of synthesis demonstrated that PmHAS added single monosaccharide units sequentially. Apparently the fidelity of the individual sugar transfer reactions is sufficient to generate the authentic repeating structure of HA. Therefore, simultaneous addition of disaccharide block units is not required as hypothesized in some recent models of polysaccharide biosynthesis. PmHAS appears distinct from other known HA synthases based on differences in sequence, topology in the membrane, and putative reaction mechanism.

Biopolymers↗
PubMed1999

Hyaluronan synthesis in virus PBCV-1-infected chlorella-like green algae.

We previously reported that the chlorella virus PBCV-1 genome encodes an authentic, membrane-associated glycosyltransferase, hyaluronan synthase (HAS). Hyaluronan, a linear polysaccharide chain composed of alternating beta1,4-glucuronic acid and beta1, 3-N-acetylglucosamine groups, is present in vertebrates as well as a few pathogenic bacteria. Studies of infected cells show that the transcription of the PBCV-1 has gene begins within 10 min of virus infection and ends at 60-90 min postinfection. The hyaluronan polysaccharide begins to accumulate as hyaluronan-lyase sensitive, hair-like fibers on the outside of the chlorella cell wall by 15-30 min postinfection; by 240 min postinfection, the infected cells are coated with a dense fibrous network. This hyaluronan slightly reduces attachment of a second chlorella virus to the infected algae. An analysis of 41 additional chlorella viruses indicates that many, but not all, produce hyaluronan during infection.

Cell Wall↗
PubMed1998

Identification and molecular cloning of a unique hyaluronan synthase from Pasteurella multocida.

Type A Pasteurella multocida, a prevalent animal pathogen, employs a hyaluronan [HA] polysaccharide capsule to avoid host defenses. We utilized transposon insertional mutagenesis to identify the P. multocida HA synthase, the enzyme that polymerizes HA. A DNA fragment from a wild-type genomic library could direct HA production in vivo in Escherichia coli, a bacterium that normally does not produce HA. Analysis of truncated plasmids derived from the original clone indicated that an open reading frame encoding a 972-residue protein was responsible for HA polymerization. This identification was confirmed by expression cloning in E. coli; we observed HA capsule formation in vivo and detected activity in membrane preparations in vitro. The polypeptide size was verified by photoaffinity labeling of the native P. multocida HA synthase with azido-UDP sugar analogs. Overall, the P. multocida sequence is not very similar to the other known HA synthases from streptococci, PBCV-1 virus, or vertebrates. Instead, a portion of the central region of the new enzyme is more homologous to the amino termini of other bacterial glycosyltransferases that produce different capsular polysaccharides or lipopolysaccharides. In summary, we have discovered a unique HA synthase that differs in sequence and predicted topology from the other known enzymes.

Amino Acid Sequence↗
PubMed1998

Enzymological characterization of recombinant xenopus DG42, a vertebrate hyaluronan synthase.

We have characterized the hyaluronan (HA) synthase activity of the Xenopus DG42 gene product in vitro. The recombinant enzyme produced in yeast does not possess a nascent HA chain and, therefore, is an ideal model system for kinetic studies of the synthase's glycosyltransferase activity. The enzymatic rate was optimal from pH 7.6 to 8.1. Only the authentic sugar nucleotide precursors, UDP-glucuronic acid (UDP-GlcA) and UDP-N-acetylglucosamine (UDP-GlcNAc), were utilized to produce a large molecular weight polymer. UDP-glucose or the galactose epimers of the normal substrates did not substitute. The Michaelis constant, Km, of recombinant DG42 in membranes was 60 +/- 20 and 235 +/- 40 microM for UDP-GlcA and UDP-GlcNAc, respectively, which is comparable to values obtained previously from membranes derived from vertebrate cells. The apparent energy of activation for HA elongation is about 15 kilocalories/mol. DG42 polymerizes HA at average rates of about 80 to 110 monosaccharides/s in vitro. The resulting HA polysaccharide possessed molecular weights spanning 2 x 10(6)-10(7) Da, corresponding to about 10(4) sugar residues. This is the first report characterizing a defined eukaryotic enzyme that can produce a glycosaminoglycan.

Animals↗
PubMed1998

Transposon Tn916 insertional mutagenesis of Pasteurella multocida and direct sequencing of disruption site.

The transposon Tn916, when introduced into Pasteurella multocida by electroporation on a nonreplicating plasmid, integrates into the bacterial chromosome. Efficiencies of approximately 8x10(4) mutants/microg of plasmid DNA were obtained. Restriction digestion and Southern analysis indicate that the Tn916 element integrates in a quasi-random fashion throughout the genome. Most transformants had a single copy of the transposon but approximately 5% had two copies. Furthermore, the nucleotide sequence at the disruption site of any desired mutant was obtained by capitalizing on the differential sensitivity of the transposon and the genome to the restriction enzyme HhaI; molecular cloning or amplification by polymerase chain reaction was not required. The Tn916 element has a single HhaI site. On the other hand, this restriction enzyme frequently cleaves the P. multocida chromosome with the vast majority of the resulting genomic fragments being less than 7 kb in length. Tn916 integration adds a 12 kb segment to the genomic HhaI fragment at the site of disruption. The resulting chimeric DNA fragment was isolated on the basis of size from digests of mutant genomic DNA separated on agarose gels. DNA sequencing with primers corresponding to the terminus of the Tn916 element was used to determine the sequence at the disruption site. In summary, Tn916 can be used to disrupt and to clone genes of P. multocida in a rapid and facile fashion.

DNA Mutational Analysis↗
PubMed1997

Hyaluronan synthase of chlorella virus PBCV-1.

Sequence analysis of the 330-kilobase genome of the virus PBCV-1 that infects a chlorella-like green algae revealed an open reading frame, A98R, with similarity to several hyaluronan synthases. Hyaluronan is an essential polysaccharide found in higher animals as well as in a few pathogenic bacteria. Expression of the A98R gene product in Escherichia coli indicated that the recombinant protein is an authentic hyaluronan synthase. A98R is expressed early in PBCV-1 infection and hyaluronan is produced in infected algae. These results demonstrate that a virus can encode an enzyme capable of synthesizing a carbohydrate polymer and that hyaluronan exists outside of animals and their pathogens.

Amino Acid Sequence↗
PubMed1996

Yeast-derived recombinant DG42 protein of Xenopus can synthesize hyaluronan in vitro.

We demonstrate in this report that the Xenopus DG42 gene product made in the yeast Saccharomyces cerevisiae can synthesize authentic high molecular weight hyaluronan (hyaluronic acid; HA) in vitro. Saccharomyces are eukaryotes that do not naturally produce HA or any other molecules known to contain glucuronic acid. Therefore bakers' yeast is a good model system to determine the enzymatic activity of the DG42 protein, which is the topic of recent debate. Membrane extracts prepared from cells expressing DG42 encoded on a plasmid incorporated [14C]glucuronic acid and N-[3H]acetylglucosamine from exogenously supplied UDP-sugar nucleotides into a high molecular mass (10(6) to 10(7) Da) polymer in the presence of magnesium ions. Both sugar precursors were simultaneously required for elongation. Control extracts prepared from cells with the vector plasmid alone or the DG42 cDNA in the antisense orientation did not display this activity. The double-labeled polysaccharide product synthesized in vitro was deemed to be HA by enzymatic analyses; specific HA lyase could degrade the polymer, but it was unaffected by protease or chitinase treatments. The fragments generated by HA lyase were identical to fragments derived from authentic vertebrate HA as compared by high performance liquid chromatography. We conclude that DG42 is a membrane-associated HA synthase capable of transferring both glucuronic acid and N-acetylglucosamine groups.

Amino Acid Sequence↗
PubMed1996

Enzymological characterization of the Pasteurella multocida hyaluronic acid synthase.

Hyaluronic acid (HA), a linear polysaccharide composed of alternating glucuronic acid and N-acetylglucosamine residues, is an essential molecule of higher vertebrates. The fowl cholera pathogen Pasteurella multocida Carter Type A also produces HA in the form of an extracellular capsule in order to evade host defenses. HA synthase activity could be obtained from cell-free membrane preparations of P. multocida. The enzyme utilized UDP-sugar precursors of HA in the presence of Mg2+ or Mn2+ at neutral pH. Mn2+ at 1 mM stimulated approximately 2-fold more incorporation than Mg2+ at 10 mM. On the other hand, the analogous enzyme from group A Streptococcus, HasA, is stimulated more by Mg2+ than Mn2+. The apparent Michaelis constants, K(M), of the P. multocida HA synthase for UDP-N-acetylglucosamine and UDP-glucuronic acid were estimated to be approximately 75 and approximately 20 microM, respectively, in the presence of Mg2+, which suggests that the substrates are bound with 2-3-fold higher affinity than by the HasA enzyme. The rate enhancement observed with Mn2+ is apparently not due to better binding of the sugar nucleotide precursors complexed to Mn ion because the K(M) value, a measure of substrate affinity, increases by 25-50% in comparison to Mg2+. In summary, the HA synthase from P. multocida, a Gram-negative bacterium, has kinetic optima distinct from those of HasA, the analog from the Gram-positive group A Streptococcus.

Cell Membrane↗
PubMed1995

Molecular cloning of the gene encoding RecF, a DNA repair enzyme, from Streptococcus pyogenes.

RecF is a component of one of the bacterial DNA repair systems. The start codon of the recF gene of Streptococcus pyogenes resides about 4.4 kb downstream from the end of hasA, the hyaluronan synthase-encoding gene, which is transcribed in the opposite orientation. The deduced 368-amino-acid RecF protein is 43.5% identical to its homolog from Bacillus subtilis. When cloned on a plasmid, recF could complement a S. pyogenes deletion mutant that was sensitive to ultraviolet irradiation and chemical mutagens. This is the first report of a recF sequence from a Gram+ pathogen.

Amino Acid Sequence↗
PubMed1994

Immunochemical confirmation of the primary structure of streptococcal hyaluronan synthase and synthesis of high molecular weight product by the recombinant enzyme.

We have recently identified and cloned the gene for hyaluronan (HA) synthase, hasA, from group A Streptococci [DeAngelis, P.L., Papaconstantinou, J., & Weigel, P.H. (1993) J. Biol. Chem. 268, 19181-19184]. We have now generated two polyclonal monospecific antibodies against synthetic peptides corresponding to portions of the deduced protein. Both antibodies recognize a protein with an apparent molecular weight of 42,000 either from wild-type Streptococcus pyogenes or from Escherichia coli containing the cloned gene on a plasmid. Immobilized affinity-purified antibody depleted HA synthase activity from functional detergent extracts of streptococcal membranes in a specific fashion. The immobilized protein displayed HA synthase activity, and HasA was the major bound polypeptide. The recombinant HA synthase behaves identically to that from Streptococci, with respect to sugar nucleotide specificity and polysaccharide production. Only the authentic sugar nucleotides UDP-glucuronic acid and UDP-N-acetylglucosamine support HA polymerization. The recombinant enzyme elongates HA in a processive manner and rapidly produces polymers on the order of > or = 5 x 10(6) Da at rates of about 10-30 monosaccharides/s at three times the apparent Km of substrates.

Amino Acid Sequence↗
PubMed1994

The Streptococcus pyogenes hyaluronan synthase: sequence comparison and conservation among various group A strains.

We recently cloned the hyaluronan synthase gene (hasA) from Streptococcus pyogenes (DeAngelis et al., J. Biol. Chem. 268, 19181, 1993). Since this is the first glycosaminoglycan synthase gene to be cloned and these enzymes have also not been purified, nothing is yet known at the molecular level about the similarity or relatedness of hyaluronan synthase to other gene products. We found several proteins in the sequence database with substantial similarities to hyaluronan synthase (HasA), including NodC from Rhizobium, DG42 from Xenopus, and the three chitin synthases (Chs) from Saccharomyces. Like HasA, NodC and the Chs proteins all have at least an N-acetylglucosaminyl transferase activity in common and are membrane-associated. The polymerase chain reaction was also used to show that HasA, and probably the two gene operon for hyaluronan biosynthesis (hasA/hasB), is highly conserved among Group A streptococcal strains. In nine strains, isolated from 1917 through 1993, only five silent mutations were detected. The results show that hyaluronan synthase is highly conserved within Group A strains, although another virulence factor, the M protein, varies considerably in these same strains.

Amino Acid Sequence↗