Liquid chromatography-mass spectrometry to study chondroitin lyase action pattern

Abstract

Liquid chromatography-mass spectrometry was applied to determine the action pattern of different chondroitin lyases. Two commercial enzymes, chondroitinase ABC (Proteus vulgaris) and chondroitinase ACII (Arthrobacter aurescens), having action patterns previously determined by viscosimetry and gel electrophoresis were first examined. Next, the action patterns of recombinant lyases, chondroitinase ABC from Bacteroides thetaiotaomicron (expressed in Escherichia coli) and chondroitinase AC from Flavobacterium heparinum (expressed in its original host), were examined. Chondroitin sulfate A (CS-A, also known as chondroitin-4-sulfate) was used as the substrate for these four lyases. Aliquots taken at various time points were analyzed. The products of chondroitinase ABC (P. vulgaris) and chondroitinase AC (F. heparinum) contained unsaturated oligosaccharides of sizes ranging from disaccharide to decasaccharide, demonstrating that both are endolytic enzymes. The products afforded by chondroitinase ABC (B. thetaiotaomicron) and chondroitinase ACII (A. aurescens) contained primarily unsaturated disaccharide. These two exolytic enzymes showed different minor products, suggesting some subtle specificity differences between the actions of these two exolytic lyases on chondroitin sulfate A.

Figure 1

Structure of CS and its cleavage by chondroitin lyase through various action patterns. A. The chemical structure of the major saccharide residues found CS-A, CS-B and CS-C, where n = 20–60. The symbolic representation of these structures is also shown with □ as GalNAc, as GlcA, and as IdoA. The predominant sequence of CS-A, CS-B and CS-C are indicated. B. A single, typical chain of CS-A is shown with some sequence heterogeneity. The non-reducing end (nre) and reducing end (re) of the chain are indicated. An exolytic enzyme cuts the chain at each cleavage site starting from the nre while a random endolytic enzyme cuts the chain through the random selection of sites, typical product distribution at time points 1 (early reaction), 2 (intermediate reaction) and ∞ (reaction completion) are shown. The symbol corresponds to unsaturated uronic acid.

Figure 2

Extracted ion chromatography (EIC) of products digested by chondroitinase ABC (P. vulgaris). A. 0 min aliquots; B. 10 min aliquots; C. 30 min aliquots; D. 60 min aliquts; and E. 120 min aliquots. Inserts are 5 × intensity magnification of 40–60 mins.

Figure 3

Mass spectra of oligosaccharides observed in Figure 2. A. Mass spectrum of dp2; B. Mass spectrum of dp4; C. Mass spectrum of dp6; D. Mass spectrum of dp8; and E. Mass spectrum of dp10.

Figure 4

EIC of products digested by chondroitinase AC II (A. aurescens). A. 0 min aliquots; B. 10 min aliquots; C. 30 min aliquots; D. 60 min aliquots; and E. 120 min aliquots. Inserts are 100X intensity magnification of 5–10 mins and 25 × intensity magnification of 20–40 mins.

Figure 5

Mass spectra of oligosaccharides observed in Figure 4. A. Mass spectrum of non-sulfated unsaturated dp2; B. Mass spectrum of mono-sulfated saturated dp2; C. Mass spectrum of mono-sulfated unsaturated dp2; D. Mass spectrum of disulfated unsaturated dp2; and E. Mass spectrum of unsaturated dp4.

Figure 6

EIC of products digested by chondroitinase ABC (B. thetaiotaomicron). A. 0 h aliquots; B. 1 h aliquots; C. 5 h aliquots; D. 12 h aliquots; and E. 24 h aliquots. Inserts are 50 × intensity magnification of 5–10 mins and 50 × intensity magnification of 20–40 mins.

Figure 7

EIC of products digested by chondroitinase AC (F. heparinum). A. 0 min aliquots; B. 10 min aliquots; C. 30 min aliquots; D. 60 min aliquots; and E. 120 min aliquots. Inserts are 2.5 × intensity magnification of 40–60 mins.