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Unbinding of hyaluronan accelerates the enzymatic activity of bee hyaluronidase.

ABSTRACT: Hyaluronan (HA), a polymeric glycosaminoglycan ubiquitously present in higher animals, is hydrolyzed by hyaluronidases (HAases). Here, we used bee HAase as a model enzyme to study the HA-HAase interaction. Located in close proximity to the active center, a bulky surface loop, which appears to obstruct one end of the substrate binding groove, was found to be functionally involved in HA turnover. To better understand kinetic changes in substrate interaction, binding of high molecular weight HA to catalytically inactive HAase was monitored by means of quartz crystal microbalance technology. Replacement of the delimiting loop by a tetrapeptide interconnection increased the affinity for HA up to 100-fold, with a K(D) below 1 nm being the highest affinity among HA-binding proteins surveyed so far. The experimental data of HA-HAase interaction were further validated showing best fit to the theoretically proposed sequential two-site model. Besides the one, which had been shown previously in course of x-ray structure determination, a previously unrecognized binding site works in conjunction with an unbinding loop that facilitates liberation of hydrolyzed HA.

SUBMITTER: Ilias A

PROVIDER: S-EPMC3195588 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Unbinding of hyaluronan accelerates the enzymatic activity of bee hyaluronidase.

Iliás Attila A Liliom Károly K Greiderer-Kleinlercher Brigitte B Reitinger Stephan S Lepperdinger Günter G

The Journal of biological chemistry 20110812 41

Hyaluronan (HA), a polymeric glycosaminoglycan ubiquitously present in higher animals, is hydrolyzed by hyaluronidases (HAases). Here, we used bee HAase as a model enzyme to study the HA-HAase interaction. Located in close proximity to the active center, a bulky surface loop, which appears to obstruct one end of the substrate binding groove, was found to be functionally involved in HA turnover. To better understand kinetic changes in substrate interaction, binding of high molecular weight HA to ...[more]

PMID: 21840987

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Project description:BackgroundPlasma hyaluronan concentrations are increased during sepsis but underlying mechanisms leading to high plasma hyaluronan concentration are poorly understood. In this study we evaluate the roles of plasma hyaluronan, effective plasma hyaluronidase (HYAL) activity and its endogenous plasma inhibition in clinical and experimental sepsis. We specifically hypothesized that plasma HYAL acts as endothelial glycocalyx shedding enzyme, sheddase.MethodsPlasma hyaluronan, effective HYAL activity and HYAL inhibition were measured in healthy volunteers (n = 20), in patients with septic shock (n = 17, day 1 and day 4), in patients with acute pancreatitis (n = 7, day 1 and day 4) and in anesthetized and mechanically ventilated pigs (n = 16). Sixteen pigs were allocated (unblinded, open label) into three groups: Sepsis-1 with infusion of live Escherichia coli (E. coli) 1 × 108 CFU/h of 12 h (n = 5), Sepsis-2 with infusion of E. coli 1 × 108 CFU/h of 6 h followed by 1 × 109 CFU/h of the remaining 6 h (n = 5) or Control with no E. coli infusion (n = 6).ResultsIn experimental E. coli porcine sepsis and in time controls, plasma hyaluronan increases with concomitant decrease in effective plasma HYAL activity and increase of endogenous HYAL inhibition. Plasma hyaluronan increased in patients with septic shock but not in acute pancreatitis. Effective plasma HYAL was lower in septic shock and acute pancreatitis as compared to healthy volunteers, while plasma HYAL inhibition was only increased in septic shock.ConclusionElevated plasma hyaluronan levels coincided with a concomitant decrease in effective plasma HYAL activity and increase of endogenous plasma HYAL inhibition both in experimental and clinical sepsis. In acute pancreatitis, effective plasma HYAL activity was decreased which was not associated with increased plasma hyaluronan concentrations or endogenous HYAL inhibition. The results suggest that plasma HYAL does not act as sheddase in sepsis or pancreatitis.

Project description:Intraventricular hemorrhage (IVH) in premature infants results in inflammation, arrested oligodendrocyte progenitor cell (OPC) maturation, and reduced myelination of the white matter. Hyaluronan (HA) inhibits OPC maturation and complexes with the heavy chain (HC) of glycoprotein inter-α-inhibitor to form pathological HA (HC-HA complex), which exacerbates inflammation. Therefore, we hypothesized that IVH would result in accumulation of HA, and that either degradation of HA by hyaluronidase treatment or elimination of HCs from pathological HA by HA oligosaccharide administration would restore OPC maturation, myelination, and neurological function in survivors with IVH. To test these hypotheses, we used the preterm rabbit model of glycerol-induced IVH and analyzed autopsy samples from premature infants. We found that total HA levels were comparable in both preterm rabbit pups and human infants with and without IVH, but HA receptors--CD44, TLR2, TLR4--were elevated in the forebrain of both humans and rabbits with IVH. Hyaluronidase treatment of rabbits with IVH reduced CD44 and TLR4 expression, proinflammatory cytokine levels, and microglia infiltration. It also promoted OPC maturation, myelination, and neurological recovery. HC-HA and tumor necrosis factor-stimulated gene-6 were elevated in newborns with IVH; and depletion of HC-HA levels by HA oligosaccharide treatment reduced inflammation and enhanced myelination and neurological recovery in rabbits with IVH. Hence, hyaluronidase or HA oligosaccharide treatment represses inflammation, promotes OPC maturation, and restores myelination and neurological function in rabbits with IVH. These therapeutic strategies might improve the neurological outcome of premature infants with IVH. Significance statement: Approximately 12,000 premature infants develop IVH every year in the United States, and a large number of survivors with IVH develop cerebral palsy and cognitive deficits. The onset of IVH induces inflammation of the periventricular white matter, which results in arrested maturation of OPCs and myelination failure. HA is a major component of the extracellular matrix of the brain, which regulates inflammation through CD44 and TLR2/4 receptors. Here, we show two mechanism-based strategies that effectively enhanced myelination and neurological recovery in preterm rabbit model of IVH. First, degrading HA by hyaluronidase treatment reduced CD44 and TLR4 expression, proinflammatory cytokines, and microglial infiltration, as well as promoted oligodendrocyte maturation and myelination. Second, intraventricular injection of HA oligosaccharide reduced inflammation and enhanced myelination, conceivably by depleting HC-HA levels.

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Unbinding of hyaluronan accelerates the enzymatic activity of bee hyaluronidase.

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Unbinding of hyaluronan accelerates the enzymatic activity of bee hyaluronidase.

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