An enzyme capture assay for analysis of active hyaluronan synthases

Hyaluronan (HA), the only non-sulfated glycosaminoglycan, is involved in morphogenesis, wound healing, inflammation, angiogenesis, and cancer. In mammals, HA is synthesized by three homologous HA synthases, HAS1, HAS2, and HAS3, that polymerize the HA chain using UDP-glucuronic acid and UDP-N-acetylglucosamine as precursors. Since the amount of HA is critical in several pathophysiological conditions, we developed a non-radioactive assay for measuring the activity of HA synthases (HASs) in eukaryotic cells and addressed the question of HAS activity during intracellular protein trafficking. We prepared three cellular fractions: plasma membrane, cytosol (containing membrane proteins mainly from the endoplasmic reticulum and Golgi), and nuclei. After incubation with UDP-sugar precursors, newly synthesized HA was quantified by polyacrylamide gel electrophoresis of fluorophore-labeled saccharides and high performance liquid chromatography. This new method measured HAS activity not only in the plasma membrane fraction but also in the cytosolic membranes. This new technique was used to evaluate the effects of 4-methylumbeliferone, phorbol 12-myristate 13-acetate, interleukin 1␤, platelet-derived growth factor BB, and tunicamycin on HAS activities. We found that HAS activity can be modulated by post-translational modification, such as phosphorylation and N-glycosylation. Interestingly, we detected a significant increase in HAS activity in the cytosolic membrane fraction after tunicamycin treatment. Since this compound is known to induce HA cable structures, this result links HAS activity alteration with the capability of the cell to promote HA cable formation.

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“…While we were working on this project, another method to assay HAS activity without using radioisotopes was developed and published (63).…”

Section: Discussionmentioning

confidence: 99%

Modulation of Hyaluronan Synthase Activity in Cellular Membrane Fractions

Vigetti

Genasetti

Karousou

et al. 2009

Journal of Biological Chemistry

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“…63. 2 ϫ 10 6 HeLa cells were transfected for 40 h and incubated with fresh medium 2 h prior to harvesting cells and lysis in 1.2 ml of lysis buffer (25 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM MgCl 2 , 0.5% Nonidet P-40, and protease inhibitor mixture) on the plate.…”

Section: Methodsmentioning

confidence: 99%

“…To examine this, HeLa cells were transfected with the indicated plasmids, and an enzyme capture assay was designed to determine enzymatically active HAS1s bound to elongating HA (Fig. 2B); an active HAS1 is expected to be bound to its polymerizing end of HA and thus can be captured with bHAPB (63), pulled down with streptavidin beads, and, as a control, treated with PBS or with HAase to remove bound enzyme from the bead complex. As demonstrated in Fig.…”

Section: Expression Of Full-length Has1 and Its Variants-humanmentioning

confidence: 99%

Aberrant Splice Variants of HAS1 (Hyaluronan Synthase 1) Multimerize with and Modulate Normally Spliced HAS1 Protein

Ghosh

K²,

Pilarski

2009

Journal of Biological Chemistry

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Most human genes undergo alternative splicing, but aberrant splice forms are hallmarks of many cancers, usually resulting from mutations initiating abnormal exon skipping, intron retention, or the introduction of a new splice sites. We have identified a family of aberrant splice variants of HAS1 (the hyaluronan synthase 1 gene) in some B lineage cancers, characterized by exon skipping and/or partial intron retention events that occur either together or independently in different variants, apparently due to accumulation of inherited and acquired mutations. Cellular, biochemical, and oncogenic properties of fulllength HAS1 (HAS1-FL) and HAS1 splice variants Va, Vb, and Vc (HAS1-Vs) are compared and characterized. When co-expressed, the properties of HAS1-Vs are dominant over those of HAS1-FL. HAS1-FL appears to be diffusely expressed in the cell, but HAS1-Vs are concentrated in the cytoplasm and/or Golgi apparatus. HAS1-Vs synthesize detectable de novo HA intracellularly. Each of the HAS1-Vs is able to relocalize HAS1-FL protein from diffuse cytoskeleton-anchored locations to deeper cytoplasmic spaces. This HAS1-Vs-mediated relocalization occurs through strong molecular interactions, which also serve to protect HAS1-FL from its otherwise high turnover kinetics. In co-transfected cells, HAS1-FL and HAS1-Vs interact with themselves and with each other to form heteromeric multiprotein assemblies. HAS1-Vc was found to be transforming in vitro and tumorigenic in vivo when introduced as a single oncogene to untransformed cells. The altered distribution and half-life of HAS1-FL, coupled with the characteristics of the HAS1-Vs suggest possible mechanisms whereby the aberrant splicing observed in human cancer may contribute to oncogenesis and disease progression.About 70 -80% of human genes undergo alternative splicing, contributing to proteomic diversity and regulatory complexities in normal development (1). About 10% of mutations listed so far in the Human Gene Mutation Database (HGMD) of "gene lesions responsible for human inherited disease" were found to be located within splice sites. Furthermore, it is becoming increasingly apparent that aberrant splice variants, generated mostly due to splicing defects, play a key role in cancer. Germ line or acquired genomic changes (mutations) in/around splicing elements (2-4) promote aberrant splicing and aberrant protein isoforms.Hyaluronan (HA) 3 is synthesized by three different plasma membrane-bound hyaluronan synthases (1, 2, and 3). HAS1 undergoes alternative and aberrant intronic splicing in multiple myeloma, producing truncated variants termed Va, Vb, and Vc (5, 6), which predicted for poor survival in a cohort of multiple myeloma patients (5). Our work suggests that this aberrant splicing arises due to inherited predispositions and acquired mutations in the HAS1 gene (7). Cancer-related, defective mRNA splicing caused by polymorphisms and/or mutations in splicing elements often results in inactivation of tumor suppressor activity (e.g. HRPT2 (8, 9), PTEN (10), MLHI (11-1...

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“…S. equi equi have a great variety of virulence factors that interact with host tissues and components which favor bacteria colonization and growth [30].The virulence factors are listed as following: Hyaluronic acid capsule: S. equi equi has the hyaluronic acid capsules which have high molecular weight polimer formed by residues of N-acetyl glucosamine and glucuronic acid. The importance of this capsule is to reduce contact of bacteria with neutrophils and subsequently phagocytosis [54]. Moreover, capsule can increase the negative charge and hydro phobicity of the bacteria surface, generating a reducing environment that protects the oxygen labile proteases and toxins [55].…”

Section: The Virulence Factor For Stranglementioning

confidence: 99%

Systematic Review: Effect of Strangle on Health of Equine Animals and its Prevention

Abdisa

2018

CTBEB

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Strangle is an infectious and highly contagious respiratory disease of equine which have great impact on the health of equnines. It is caused by streptococcus equi sub sp. equi, a gram positive bacterium in irregular shaped cocci that forms long chain. Strangles occurs worldwide mostly in horses and rarely in donkey and mules. The outbreak of strangle is frequently seen on racing stable, riding and breeding farms when the infection is introduced by new arrivals that are often carrier. Strangle disease affects mostly young horses and outbreak can occur at any time of the year but, most likely to happen in cold weather. The severity of the clinical presentation depends on age, previous infections, vaccination and stabling conditions. Strangle can be transmitted to other equine animals buy direct and in direct ways. The causative agent of strangle, S. equi var equi have great variety of virulence factors that interact with host tissues and components, and favour bacteria colonization and growth. The pathogenesis of strangle disease is begin with nasopharyngeal infection with S. equi var equi follows contact with a shedding carrier horse. The after the bacteria begin to multiply in lymph nodes.The clinical sign which observed from diseased animal are like complete anorexia, depression, fever, serous nasal discharge which becomes copious and mucopurulent, loss of body condition and enlargement of Sub mandibular lymph node with subsequent dyspnea. The diagnosis of strangle disease is based on clinical sign and laboratory finding including bacterial culture, PCR and serology. The treatment of diseased animal in the early stage is usually effective, but it is difficult to treat which are in chronic stage of disease. The best way to prevent an outbreak of strangle disease is by keeping hygiene and giving vaccination for equine animals. Therefore, the objective of this manuscript is to overview general fact of strangle disease and how it effect on the health of equine animal. And also it give enough information for the future study on the strangle disease in species of equine animals.

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An enzyme capture assay for analysis of active hyaluronan synthases

Cited by 4 publications

References 34 publications

Modulation of Hyaluronan Synthase Activity in Cellular Membrane Fractions

Modulation of Hyaluronan Synthase Activity in Cellular Membrane Fractions

Aberrant Splice Variants of HAS1 (Hyaluronan Synthase 1) Multimerize with and Modulate Normally Spliced HAS1 Protein

Systematic Review: Effect of Strangle on Health of Equine Animals and its Prevention

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