Bacterial glycosaminoglycan lyases to depolymerize hyaluronan

Authors：

Category

Glycosaminoglycans

Protocol Name

Authors

Yamada, Shuhei
Department of Pathobiochemistry, Faculty of Pharmacy, Meijo University

KeyWords

chondroitinase anion-exchange chromatography hyaluronidase

Reagents

●	HA preparation having an average molecular mass of 35 kDa produced by microbial fermentation of Streptococcus pyogenes (R & D Systems, Minneapolis, MN)
●	Hyaluronidase from Streptomyces hyalurolyticus (Seikagaku Biobusiness Corp., Tokyo, Japan)
●	Hyaluronidase SD from Streptococcus dysgalactiae (Seikagaku Biobusiness Corp., Tokyo, Japan)
●	Chondroitinase ABC (a conventional preparation) from Proteus vulgaris (Seikagaku Biobusiness Corp., Tokyo, Japan)
●	Chondroitinase AC-I from Flavobacterim heparinum (Seikagaku Biobusiness Corp., Tokyo, Japan)
●	Chondroitinase AC-II from Arthrobacter aurescens (Seikagaku Biobusiness Corp., Tokyo, Japan)

Instruments

●	YMC-Pack PA-03 (4.6 mm i.d. x 250mm) (YMC Co., Ltd., Kyoto, Japan)
●	HPLC system (LC-20 Series; Shimadzu Corp. Kyoto, Japan)
●	Ultrafree-MC (Merck Millipore, Billerica, MA)

Methods

Bacterial glycosaminoglycan lyases to depolymerize hyaluronan

1)	Enzymatic digestion of HA a) Incubate HA (2 μg) with hyaluronidase from Streptomyces hyalurolyticus (0.5 TRU) in a total volume of 20 μL of 20 mM acetate-Na buffer, pH 6.0, containing 150 mM sodium chloride at 60˚C for 75 min or hyaluronidase SD from Streptococcus dysgalactiae (5 mIU) in a total volume of 20 μL of 40 mM phosphate-Na buffer, pH 6.2, at 37˚C for 30 min. b) Incubate HA (2 μg) with chondroitinase (5 mIU each) in a total volume of 20 μL of 50 mM Tris-HCl buffer, pH 8.0, containing 60 mM sodium acetate (chondroitinase ABC), 50 mM Tris-HCl buffer, pH 7.3 (chondroitinase AC-I), or 50 mM acetate-Na buffer, pH 6.0 (chondroitinase AC-II), at 37˚C for 30 min.	Comment 1

2)	Terminate the reactions by boiling at 100˚C for 1 min.	Comment 0

3)	Dilute the reaction mixture with distilled water to 200 μL.	Comment 0

4)	Analyze an aliquot (100 μL) by HPLC on an amine-bound silica PA-03 column using a linear gradient of NaH₂PO₄ from 16 to 530 mM at a flow rate of 1 mL/min, monitored by measuring UV with a wavelength of 232 nm.	Comment 0

Identify and quantify the eluted peaks by comparing with the positions of authentic standards (Fig. 1 and 2)

Comment 1

Figure & Legends

Enzyme	Substrate Specificity	Predominant End Products
Hyaluronidase from Streptomyces hyalurolyticus	This enzyme is quite specific for HA, and acts on neither chondroitin sulfate (CS) nor chondroitin.	Unsaturated tetrasaccharide and hexasaccharide
Hyaluronidase SD from Streptococcus dysgalactiae	This enzyme acts on not only HA but also chondroitin. However, it does not depolymerize CS. The rate of degradation of chondroitin is much slower than that of HA.	Unsaturated disaccharide
Chondroitinase ABC from Proteus vulgaris	HA is hardly degraded by this enzyme. It acts on both CS and chondroitin, but the rate of depolymerization of chondroitin is slower than that of CS.	Unsaturated disaccharide
Chondroitinase AC-I from Flavobacterim heparinum	This enzyme depolymerizes CS and chondroitin at a similar rate. It also degrades HA, though slightly slower than CS/chondroitin.	Unsaturated disaccharide
Chondroitinase AC-II from Arthrobacter aurescens	This enzyme acts on not only CS/chondroitin but also HA in an exolytic fashion.	Unsaturated disaccharide

Table 1. Commercially available bacterial hyaluronidases as well as chondroitinases

Treatment with bacterial eliminases converts the original uronic acid structure in the polysaccharides into an artificial structure, the 4,5-unsaturated uronic acid, 4-deoxy-α-L-threo-hex-4-enepyranosyluronic acid.

Fig. 1. HPLC profiles of the hyaluronidase digests.

The HA preparation (2 μg) was exhaustively digested with the hyaluronidase from Streptomyces hyalurolyticus (A) or hyaluronidase SD from Streptococcus dysgalactiae (B). Digests were analyzed by anion-exchange HPLC on an amine-bound silica column using a linear gradient of NaH₂PO₄ from 16 mM to 530 mM over 60 min. Eluates were monitored by UV-absorbance at 232 nm. Arrows indicate the elution positions of authentic unsaturated disaccharides. a, ΔHexA-GlcNAc and ΔHexA-GalNAc; b, ΔHexA-GalNAc(6-sulfate); c, ΔHexA-GalNAc(4-sulfate); d, ΔHexA(2-sulfate)-GalNAc(6-sulfate); e, ΔHexA-GalNAc(4,6-disulfate). ΔHexA, GalNAc, and GlcNAc represent 4,5-unsaturated uronic acid, N-acetylgalactosamine, and N-acetylglucosamine, respectively. Presumable positions of unsaturated oligosaccharides are shown by open arrows. f, unsaturated tetrasaccharide: g, unsaturated hexasaccharide: h, unsaturated octasaccharide: i, unsaturated decasaccharide.

Fig. 2. HPLC profiles of the chondroitinase digests.

The HA preparation (2 μg, approximately 5 nmol as disaccharide) was digested with 5 mIU of chondroitinase ABC (A), AC-I (B), or AC-II (C) at 37˚C for 30 min. Reactions were terminated by heat treatment at 100˚C for 1 min. Digests were analyzed by anion-exchange HPLC on an amine-bound silica column, being monitored by measuring UV-absorbance at 232 nm. Arrows in panel A indicate the elution positions of authentic unsaturated disaccharides (see the legend to Fig. 1). The open arrow f in panel B shows the presumable position of the unsaturated tetrasaccharide. The chromatogram in panel A was enlarged ten times to compare it with those in panels B and C. The ratio of the major unsaturated oligosaccharides generated by enzymatic digestion was calculated based on the peak area, and is indicated in the figure taking the value obtained in the digest with chondroitinase AC-II as 100.

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Date of registration：2013-12-27 12:03:18