Quantitative continuous assay for hyaluronan synthase

Abstract

A rapid, continuous, and convenient three-enzyme coupled UV absorption assay was developed to quantitate the glucuronic acid and N-acetylglucosamine transferase activities of hyaluronan synthase from Pasteurella multocida (PmHAS). Activity was measured by coupling the UDP produced from the PmHAS-catalyzed transfer of UDP-GlcNAc and UDP-GlcUA to a hyaluronic acid tetrasaccharide primer with the oxidation of NADH. Using a Xuorescently labeled primer, the products were characterized by gel electrophoresis. Our results show that a truncated soluble form of recombinant PmHAS (residues 1-703) can catalyze the glycosyl transfers in a time-and concentration-dependent manner. The assay can be used to determine kinetic parameters, inhibition constants, and mechanistic aspects of this enzyme. In addition, it can be used to quantify PmHAS during purification of the enzyme from culture media. KeywordsPasteurella multocida hyaluronan synthase; UDP-glycosyltransferase; Glycosaminoglycan; Hyaluronan synthase Glycosaminoglycans (GAGs) 1 are naturally occurring biopolymers consisting of disaccharide repeats that often are N-and/or O-sulfated. There are four main classes of GAGs: heparin and heparan sulfate, chondroitin and dermatan sulfate, keratan sulfate, and hyaluronic acid (HA). HA, which consists of the disaccharide repeat GlcUA(β1-3)-GlcNAc(β1-4), plays an important role in normal health and development [1] as well as in inflammation [2], wound repair [3], and cancer pathology [4,5]. GAGs, and in particular HA, have been used therapeutically (reviewed in Ref. [6]) in the treatment of arthritis [7] and cancer [8]. However, in some bacteria, HA forms part of the external capsule [ NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript thereby contributing to pathogenicity [10,11]. Hyaluronan synthases (HASs) are the glycosyltransferases responsible for the biosynthesis of HA. These enzymes are dual-action Materials and methods MaterialsFermentation-generated ultrapure HA was a generous gift from Clear Solutions Biotech (Stony Brook, NY, USA). HA used for the generation of the Xuorophore-assisted carbohydrate electrophoresis (FACE) HA primer was a generous gift from Toshihiko Toida (Chiba University, Japan). Size exclusion chromatography media BioGel P-2 was purchased from Bio-Rad (Hercules, CA, USA Cloning, expression, and purification of recombinant PmHASThe gene coding for PmHAS as a soluble form (residues 1-703) was amplified by standard PCR techniques from the genomic DNA of P. multocida type A strain P-1059 using the following forward and reverse primers: AAA-AAA-CAT-ATG-AAT-ACA-TTA-TCA-CAA-GCA-ATA-AAA-GC and AAA-AAA-GGA-TCC-TTA-AAT-ATC-TTT-TAA-GAT-ATC-AAT-CTC-TTC-TTG. These primers introduced NdeI and BamHI restriction sites (underlined), respectively. The resulting DNA fragment was cloned into a modified pET15b vector (Novagen, Madison, WI, USA), coding for an N-terminal His8 tag followed by a thrombin cleavage site, all under the control of the T7 promoter. The DNA was transformed into the...

Structural and functional evidence for two separate oligosaccharide binding sites of Pasteurella multocida hyaluronan synthase

“…Recently, it was demonstrated that, for Streptococcus zooepidemicus, overexpression of genes involved in UDP-GlcNAc biosynthesis increased the molecular weight of the HA products [22], indicating that the chain length is controlled by the availability of the substrates. This is corroborated by kinetic data from HAS enzymes of different sources, demonstrating a significantly larger K MNAc value than K MUA value [23][24][25][26]. Furthermore, the hyaluronan synthase in Pasteurella multocida (PmHAS) possesses the ability to elongate HA oligosaccharides [27], which offers an additional opportunity to optimize product length and polydispersity.…”

“…A coupled-enzyme assay, similar to assays created for other glycosyltransferases [23,31,32], was developed to measure PmHAS activity. The coupled-enzyme assay directly links the increase in the UDP by-product of the PmHAS elongation to the decrease of NADH that was spectrophotometrically measured at 340 nm.…”

Elastographic Assessment of Xenograft Pancreatic Tumors

“…Second, because AsPc-1 tumors are heterogeneous (Bernhaus et al 2009; Tan and Chu 1984), simple histology may not provide an accurate assessment of the amounts of collagen and hyaluronic acid within the tumors. To address this limitation, we plan to use a soluble assay to quantify the collagen and hyaluronic acid content more accurately (Barnes et al 2009; Krupa et al 2007; Nagelschmidt and Viell 1987). Third, we performed all measurements with ex vivo samples; therefore, we could not assess the effects of blood pressure on Young’s modulus.…”

In vitro synthesis of heparosan using recombinant Pasteurella multocida heparosan synthase PmHS2

“…Both nucleotide sugars (UDP-glucuronic acid and UDP-N-acetylglucosamine) were added in equimolar ratio and in sufficient amount in order to initiate the polymerization and enable us to follow the elongation process in time by means of the coupled enzyme assay and agarose gel analysis. The activity of PmHS2 was assessed by measuring the UDP formation with a noncontinuous spectrophotometric assay at 340 nm (Gosselin et al 1994 ; Krupa et al 2007 ). In this assay, the UDP released from the conversion of the nucleotide sugars during the polymerization is coupled via pyruvate kinase (PK) and lactate dehydrogenase (LDH) to the oxidation of NADH into NAD + .…”