Sequencing Complex Polysaccharides Ganesh Venkataraman, Zachary Shriver, Rahul Raman, and Ram Sasisekharan

We and others have observed secondary specificities of the heparinases, especially under exhaustive degradation conditions. As a part of ongoing investigations into the enzymology of heparinases, we have measured the relative rates of cleavage of I- and G-containing sites by heparinase I and III with defined substrates under different conditions. For instance, heparinase III cleaves both at I- and G-containing linkages and not at I_2S (1). However, under the reaction conditions used in this study, there is a dramatic (8- to 10-fold) difference in the rates of cleavage, with I-containing linkages being clipped more slowly than G-containing linkages (Web Fig. 1A). Under the "short" conditions of digest (2), it is expected that only G-containing saccharides are cleaved to an appreciable extent. For example, with the hexasaccharide DUH_NH,6SGH_NSIH_NAc (which contains both I and G in a minimally sufated region), cleavage occurs only at the G under short digest conditions (Web Fig. 1B). Furthermore, we have found that the degree of sulfation does affect the kinetics of heparinase III degradation of oligosaccharides (3). In the case of heparinase I, we find that this enzyme does not clip either I- or G-containing glycosidic linkages within the context of our experimental procedures, whereas it readily clips I2S-containing oligosaaccharides (Web Fig. 1C). There is only one report of heparinase I clipping G_2S-containing linkages (4). This was tested with two tetrasaccharide substrates, and the experiments were performed under conditions that were kinetically very different from the short heparinase I digestion presented here.

Quite a few factors have severely limited and complicated studies and interpretation of heparinase substrate specificity experiments. First, not only is preparation of a homogenous substrate difficult, but analyzing the substrates and products has been very challenging as well. Analysis has primarily relied on comigration of the saccharides with known standards; and as we and others have observed, oligosaccharides with different sulfation patterns do comigrate, complicating unique assignments. Further, some oligosaccharides used in previous studies to assign substrate specificity for the heparinases were not homogeneous, complicating analysis. The development of the MALDI-MS procedure has enabled rapid and accurate determination of the saccharides. The second problem is the preparation of pure wild-type heparinases from the native host. The wild-type heparinase is isolated from Flavobacterium heparinum. This organism produces several complex polysaccharide-degrading enzymes, and these often copurify with each other. For example, when examining the kinetics of heparinase III, we found that a commercial source of heparinase III was able to degrade the supposedly noncleavable DU_2SH_NS,6SI_2SH_NS,6S (Web Fig. 2A). Furthermore, MS and CE analysis of the products indicated that one was specifically 2-O desulfated, which suggests a sulfatase contamination. Recombinant heparinase III produced and purified in our laboratory (and not having contamination with other heparin-degrading enzymes) does not cleave DU_2SH_NS,6SI_2SH_NS,6S (Web Fig. 2B) as expected. Thus, different enzyme preparations and differences in digestion conditions, as well as differences in substrate size and composition and often contaminating substrates, taken together with assignments based on coelution, make comparison of data very difficult and also has led to contradictory findings.

Regardless of the outcome of heparinase substrate specificities, there are other methods that can be used to extract the isomeric state of the uronic acid (I or G or I_2S or G_2S). The uronic acid component of each disaccharide unit can be unambiguously ascertained by completing compositional analysis after exhaustive nitrous acid treatment, and this is widely described by many groups. By this method, compositional analysis of given oligosaccharides can be accomplished and the presence of G_2S-, I_2S-, I-. and G-containing building blocks assessed. With this information, rapid convergence to a single sequence could be completed by judicious application of the heparinases (regardless of their exact substrate specificity), because cleavage would give mass information on either side of the cleavage site. Thus, in the octasaccharide (example 1) case, application of exhaustive nitrous acid would yield 1x DUMan_6S, 2x I_2SMan_6S, and 1x GMan_6S. Then digestion of this octasaccharide, after tagging, with heparinase III under any conditions (forcing or nonforcing) would result in the formation of a hexasaccharide (m/z 5958.7) and a disaccharide, immediately fixing the sequence. A similar sequence of events can be used with heparinase I to converge to a single sequence for the octasaccharide.

Although there are caveats regarding the use of any one particular system for sequence analysis, whether the system is chemical degradation or enzymatic analysis, the sequencing strategy presented here is not critically dependent on any single technique. One of the major strengths of our sequencing strategy is the flexibility of our approach and that the integration of MALDI and the coding scheme enable us to adapt to different experimental constraints (5). As stated, additional or different sets of experimental constraints can be used to not only arrive at a unique solution but also to validate or confirm the solution from a given set of experimental constraints.

References and Notes:
1. H. E. Conrad, Heparin Binding Proteins (Academic Press, San Diego, CA, 1998).
2. Conditions for enzymatic digest of HLGAG oligosaccharides are explained in reference (18) of the published text.
3. S. Ernst et al., Crit. Rev. Biochem. Mol. Biol. 30, 387 (1995); S. Yamada et a/., Glycobiology 4, 69 (1994); U. R. Desai, H. M. Wang, R. J. Linhardt, Biochemistry 32, 8140 (1993); R. J. Linhardt et al., Biochemistry 29, 2611 (1990).
4. S. Yamada, T. Murakami, H. Tsuda, K. Yoshida, K. Sugahara, J. Biol. Chem. 270, 8696 (1995).
5. For example, the recently cloned mammalian heparinase is another possible experimental constraint [M. D. Hulett et a/., Nature Med. 5,793 (1999); I. Vlodavsky et a/., Nature Med. 5, 803 (1999)].

Web Fig. 1. (A) Cleavage by recombinant heparinase III (used in this study) of tetrasaccharides containing G (),
I (), or I_2S () linkages. Each reaction was followed by capillary electrophoresis. With these substrates, heparinase III does not cleave I_2S-containing glycosidic linkages and cleaves G-containing linkages roughly 10 times as fast as I-containing linkages. (B) Heparinase III was incubated with the hexasaccharide DUH_NH,6SGH_NSIH_NAc, and only cleavage at the G and not the I was observed. (C) Same study as completed in (A), except that heparinase I was used instead of heparinase III. With heparinase I, cleavage only occurs at I_2S-containing linkages but not before I or G.

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Web Fig. 2. (A) CE analysis of cleavage of DU_2SH_NS,6SI_2SH_NS,6S with commercially available heparinase III. 100 (M of the substrate was incubated with 1 mU of heparinase III for 24 hours at room temperature. The peak at 8.36 min coelutes with the disaccharide standard DU_2SH_NS,6S, whereas the peak at 10.5 min coelutes with the standard DUH_NS,6S, a desulfation product of the substrate. (B) Digest of DU_2SH_NS,6SI_2SH_NS,6S under identical conditions to those outlined in (A), except for the fact that recombinant heparinase III produced in Escherichia coli was used for the digest. In this case, no digestion products are observed.

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