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.
Highlights
Hyaluronan (HA)3 is an unbranched glycosaminoglycan, which is composed of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine, and functional roles thereof are related to its polymeric size [1]
Substrate samples were prepared by serial dilutions in running buffer, and the interaction was recorded with the aid of Attester® software (Attana AB) for HA concentrations ranging from 0.05 to 1.0 mg/ml and for chondroitin sulfate concentrations ranging from 0.05 to 0.5 mg/ml injected in a random order at 37 °C
Enzyme Activity and Kinetic Analysis—The substrate binding groove of HAase is constrained at one end by a bulky noose, which comprises a short stretch of amino acids that loop out at a cystine bridge linking position 189 and 201
Summary
Materials—Hyaluronic acid potassium salt from human umbilical cord with an estimated average molecular mass of 750 kDa (H1504, lot no. 097K1495), chondroitin sulfate A (27042, lot STBC0183V) with an estimated average molecular mass of 21 kDa [20, 21], chondroitin sulfate B (C3788, lot no. 080M1668V) of ϳ42 kDa [21], chondroitin sulfate C (C4384, lot no. 1426300V) of ϳ60 kDa [22], N-acetyl-glucosamine (A8625), potassium tetraborate tetrahydrate (P5754), and p-dimethyl-benzaldehyde (D2004) were purchased from Sigma. High molecular weight HA (5 mg/ml), or as a standard, N-acetyl-D-glucosamine were diluted to the desired concentration with 100 mM citrate buffer, pH 4.0, containing 137 mM NaCl and 2.7 mM KCl in a total volume of 100 l. Substrate samples were prepared by serial dilutions in running buffer, and the interaction was recorded with the aid of Attester® software (Attana AB) for HA concentrations ranging from 0.05 to 1.0 mg/ml and for chondroitin sulfate concentrations ranging from 0.05 to 0.5 mg/ml injected in a random order at 37 °C. The frequency response curves of the interaction between the various substrates at different concentrations and the immobilized inactive BVH variants, as well as the unmodified surface were analyzed by the Attache Evaluation software (version 3.3.3.1, Attana AB). The equilibrium dissociation constants of BVH variants for high molecular weight HA and chondroitin sulfates were calculated from the directly estimated association and dissociation rate constants (KD ϭ kdiss/ka)
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