Natisha L. Rose

Galactosyl Transferases in Mycobacterial Cell Wall Synthesis

Two galactosyl transferases can apparently account for the full biosynthesis of the cell wall galactan of mycobacteria. Evidence is presented based on enzymatic incubations with purified natural and synthetic galactofuranose (Galf) acceptors that the recombinant galactofuranosyl transferase, GlfT1, from Mycobacterium smegmatis, the Mycobacterium tuberculosis Rv3782 ortholog known to be involved in the initial steps of galactan formation, harbors dual beta-(1-->4) and beta-(1-->5) Galf transferase activities and that the product of the enzyme, decaprenyl-P-P-GlcNAc-Rha-Galf-Galf, serves as a direct substrate for full polymerization catalyzed by another bifunctional Galf transferase, GlfT2, the Rv3808c enzyme.

Development of a coupled spectrophotometric assay for GlfT2, a bifunctional mycobacterial galactofuranosyltransferase

As a key constituent of their protective cell wall all mycobacteria produce a large structural component, the mycolyl-arabinogalactan (mAG) complex, which has at its core a galactan moiety of alternating beta-(1-->5) and beta-(1-->6) galactofuranosyl residues. Galactan biosynthesis is essential for mycobacterial viability and thus inhibitors of the enzymes involved in its assembly are potential drugs for the treatment of mycobacterial diseases, including tuberculosis. Only two galactofuranosyltransferases, GlfT1 and GlfT2, are responsible for the biosynthesis of the entire galactan domain of the mAG and we report here the first high-throughput assay for GlfT2. Successful implementation of the assay required the synthesis of multi-milligram amounts of the donor for the enzyme, UDP-Galf, 1, which was achieved using a chemoenzymatic approach. We also describe an improved expression system for GlfT2, which provides a larger amount of active protein for the assay. Kinetic analysis of 1 and a known trisaccharide acceptor for the enzyme, 2, have been carried out and the apparent K(m) and k(cat) values obtained for the latter are in agreement with those obtained using a previously reported radiochemical assay. The assay has been implemented in 384-well microtiter plates, which will facilitate the screening of large numbers of potential GlfT2 inhibitors, with possible utility as novel anti-TB drugs.

Differential Recognition of the Type I and II H Antigen Acceptors by the Human ABO(H) Blood Group A and B Glycosyltransferases.

The human ABO(H) blood group A and B antigens are generated by the homologous glycosyltransferases A (GTA) and B (GTB), which add the monosaccharides GalNAc and Gal, respectively, to the cell-surface H antigens. In the first comprehensive structural study of the recognition by a glycosyltransferase of a panel of substrates corresponding to acceptor fragments, 14 high resolution crystal structures of GTA and GTB have been determined in the presence of oligosaccharides corresponding to different segments of the type I (α-l-Fucp-(1→2)-β-d-Galp-(1→3)-β-d-GlcNAcp-OR, where R is a glycoprotein or glycolipid in natural acceptors) and type II (α-l-Fucp-(1→2)-β-d-Galp-(1→4)-β-d-GlcNAcp-OR) H antigen trisaccharides. GTA and GTB differ in only four “critical” amino acid residues (Arg/Gly-176, Gly/Ser-235, Leu/Met-266, and Gly/Ala-268). As these enzymes both utilize the H antigen acceptors, the four critical residues had been thought to be involved strictly in donor recognition; however, we now report that acceptor binding and subsequent transfer are significantly influenced by two of these residues: Gly/Ser-235 and Leu/Met-266. Furthermore, these structures show that acceptor recognition is dominated by the central Gal residue despite the fact that the l-Fuc residue is required for efficient catalysis and give direct insight into the design of model inhibitors for GTA and GTB.

Evaluation of pefabloc as a serine protease inhibitor during human-islet isolation

Background. Recent evidence has suggested that inconsistencies in human-islet yields after collagenase digestion are attributed to the activation of endogenous enzymes of the cadaveric donor pancreas. Inhibition of protease activity by Pefabloc (0.4 mM; Roche Biochemicals Inc., Indianapolis, IN) has recently been shown to improve human-islet isolation after prolonged cold storage of the pancreas. In this study, we have hypothesized that this improvement was because of the inhibition of three key serine proteases. Methods. Twenty cadaveric pancreases were perfused in the presence (n=12) and absence (n=8) of Pefabloc added at the time of distention using a customized perfusion device. Samples were collected throughout the digestion process and were assayed for trypsin, chymotrypsin, elastase, and total protease activity. Results. In all cases, the enzyme activity levels remained lower in the presence of Pefabloc as compared with the control samples. There was significantly higher chymotrypsin and elastase activity in the control group, but not trypsin or total protease activity, from the time following loading of the enzyme onto the pancreas until the stopping of the enzymatic digestion phase (dilution). Conclusions. Pefabloc was shown to be an effective protease inhibitor throughout the entire digestion process. Pefabloc supplementation did not significantly effect the dilution time or the islet yield in this study; however, these data show that serine proteases are effectively inhibited by Pefabloc during the clinical islet process.

Endogenous Pancreatic Enzyme Activity Levels Show no Significant Effect on Human Islet Isolation Yield

Despite advances in human islet isolation, islet yield remains inconsistent and unreliable. In recent studies, it has been suggested that serine proteases, in particular trypsin, have been shown to have a damaging effect on islet yield. This study evaluated enzyme activity levels throughout 42 human islet isolation procedures. Trypsin, chymotrypsin, and elastase activity was determined spectrophotometrically using suitable chromophoric substrates. The results of the islet isolations were rated as successful (n = 19) or unsuccessful (n = 23) based on the islet yield and functionality. The enzyme activity profiles of the isolations were compared. No significant differences in donor-related variables were found in this study. However, in the successful isolations, a significantly greater amount (85.6 ± 1.9%; p = 0.0017) of the pancreas was digested in a significantly shorter digestion time (19.7 ± 0.6 min; p = 0.0054) compared with 74.8 ± 2.5% of digested tissue in 22.6 ± 0.7 min in the poor isolations. This study showed no significant effect of serine protease levels on the outcome of islet isolations, regardless of enzyme inhibitor supplementation. These data suggest that serine protease activity does not sufficiently affect islet yield. However, the data show that the most successful human islet isolations are achieved when the maximum amount of tissue is digested in the shortest amount of time. This suggests that further understanding of the isolation process should focus on the role of the collagenase digestion solution in the dissociation of the endocrine–exocrine tissue connection.

Evaluating the effect of serine proteases on collagenase activity during human islet isolation.

Inconsistencies in human islet yields after collagenase digestion have been attributed to the activation of endogenous enzymes of the donor pancreas. It has been suggested that pancreatic serine proteases contribute to the proteolysis of collagenase. This study defined the effects of endogenous enzymes within the pancreas on pancreas dissociation during collagenase digestion. Levels of collagenase activity from samples taken throughout several steps in islet isolation procedures, both with and without the addition of the serine protease inhibitor Pefabloc, were determined by a spectrophotometric assay using N-[3-(2-furyl)acryloyl]-Leu-Gly-Pro-Ala as the substrate. Results clearly demonstrated that the level of collagenase activity remains stable throughout the isolation procedure despite differences in the donor factors from several cadaveric donor pancreases. This was further demonstrated by observing no difference in activity levels after incubating commercial collagenase preparations with serine proteases and analyzing by means of collagenase activity and SDS-PAGE. These data show that the presence of serine proteases does not affect the level of collagenase activity; however, they likely damage the islet cells upon prolonged digestion of the pancreatic tissue. Further efforts at examining exogenous and endogenous enzyme levels may result in the development of an enzyme cocktail that is both stable and effective for digesting the human pancreas while preserving islet function and viability.

Amino-acid substitution in the disordered loop of blood group B-glycosyltransferase enzyme causes weak B phenotype

BACKGROUND: Few studies have investigated the reaction kinetics and interactions with nucleotide donor and acceptor substrates of mutant human ABO glycosyltransferases. Previous work identified a Bw allele featuring a 556G>A polymorphism giving rise to a weak B phenotype. This polymorphism is predicted to cause a M186V amino‐acid mutation within a highly conserved series of 16 amino acids present both in both blood group A– and blood group B–synthesizing enzymes. These residues are known as the disordered loop because their location cannot be determined in the crystal structure of the enzyme. Another patient has been identified with a 556G>A Bw allele and the kinetics of the resulting mutant glycosyltransferase were studied.

An Evaluation of Endogenous Pancreatic Enzyme Levels after Human Islet Isolation

Introduction Recent evidence has suggested that inconsistencies in human islet yield and viability after collagenase digestion is attributed to the activation of endogenous enzymes of the cadaveric donor pancreas. A study of the enzyme kinetics of serine proteases throughout human islet isolations showed a significant increase in activity levels throughout the digestion period. Following the digestion, it is important to further inhibit these enzymes by the addition of an inhibitor to the dilution media. Aim To report the levels of endogenous pancreatic enzymes remaining after human islet isolation and the effects of three potential enzyme inhibitors on the proteases. Methodology Human albumin, fetal calf serum, and the protease inhibitor aprotinin were incubated with the trypsin, chymotrypsin, elastase, and collagenase and were assayed for activity. Results Results at the final stage indicated that chymotrypsin retained 21.0 ± 7.5% (mean ± SE; n = 20) of the activity observed at the conclusion of the enzymatic digestion phase of the isolation process, whereas trypsin, elastase, and collagenase retained 3.0 ± 1.5%, 2.1 ± 0.6%, and 3.9 ± 0.9%, respectively. Fetal calf serum and aprotinin showed strong inhibitory effects against bovine pancreatic trypsin; however, they showed a weak inhibitory effect against elastase. Supplementation with aprotinin failed to inhibit human chymotrypsin and elastase. Human albumin showed minimal inhibition and was shown to serve only as a competitive inhibitor. No inhibition to collagenase was observed with human albumin, fetal calf serum, or aprotinin. Conclusions This study clearly demonstrates that low amounts of endogenous pancreatic enzymes remain active throughout the human islet isolation process and that the added inhibitors at the end of the isolation process are not fully effective at inhibiting the enzymes.

Kinetic studies on Korean serum cis‐AB enzymes reveal diminished A and B transferase activities

Background  cis‐AB enzymes are rare glycosyltransferases that synthesize both blood group A and B antigens. We have identified a large cohort of Korean cis‐AB blood donors and studied the N‐acetylgalactosaminyltransferase (glycosyltransferase A, GTA) and galactosyltransferase (glycosyltransferase B, GTB) activity of their cis‐AB serum enzymes.