A-glycanase

In order to assess the size of the carbohydrate component, deglycosylated CHO IL-4 by A-glycanase was analyzed by ESI-MS. The deconvoluted spectrum displayed an MW of 14,955 Da, which conformed well to the theoretical MW of the protein (MW 14,963 Da) when considering the presence of three disulfide bonds in the protein. 

Suzuki, T., Park, H., Kitajima, K., and Lennarz, W. J. (1998). Peptides glycosylated in the endoplasmic reticulum of yeast are subsequently deglycosylated by a soluble peptide A-glycanase activity. J. Biol. Chem. 273, 21526-21530. 

Lerouge, P., O Neill, M.A., Darvill, A.G., and Albersheim, P. (1993) Structural characterization of endo-glycanase-generated oligoglycosyl side chains of rhamnogalactuTonan I. CarbohydrMes. 243 359-371. 

For the purification of RHG, which has been described before (6), A. aculeatus was grown in sugar-beet pulp medium for 48 h. From a 2 1 culture containing 3.2 g protein, about 5 mg RHG was obtained after purification. Analysis by SDS-PAGE revealed only one band of about 55 kDa and thus the enzyme appeared to be pure. After N-glycanase treatment and SDS-PAGE analysis, a smaller protein band of about 46 kDa was visible (Fig.l). 

The same principle described above to monitor endo-peptidase activity was used by Matsuoka to monitor a ceramide glycanase... 

Matsuoka, K., Nishimura, S. I. and Lee, Y. C. (1995). A bi-fluorescence-labeled substrate for ceramide glycanase based on fluorescence energy-transfer. Carbohydr. Res. 276, 31-42. 

Nishimura and Yamada [10-11] introduced a water-soluble polymeric support having a linker recognized by ceramide glycanase for a synthesis of ganghoside GM3 (17). Synthesis of the polymerizable lactose derivative (14) with a ceramide glycanase sensitive linker is shown in Scheme 10.3. The lactosyl ceramide (Lac-Ger) mimetic glycopolymer (15) is obtained from the monomeric precursor (14) by co-polymerization with acrylamide. 

Scheme 10.3 Ceramide glycanase mediated release by transglycosylation. a) Pd/C, MeOH b) (8), EEDQ, EtOH-CgHe c) MeONa d) CH2=CHCONH2, TMEDA, APS, DMSO-H2O, 50"C, CMP-NeuAc, a-2,3-sialyltransferase, BSA, MnCb, CIAP, 50 nM sodium cacodylate buffer, pH 7.49, ceramide, Triton CF-54, sodium citrate buffer, pH 6.0, 37°C, 61%.

Suzuki T, Seko A, Kitajima K, Inoue Y, Inoue S (1994) Purification and enzymatic properties of peptide N-glycanase from C3H mouse-derived L-929 fibroblast cells Possible widespread occurrence of post-translational remodification of proteins by N-deglycosylation. J Biol Chem 269 17611-17618 Swaminathan S, Amerik AY, Hochstrasser M (1999) The Doa4 deubiquitinating enzyme is required for ubiquitin homeostasis in yeast. Mol Biol Cell 10 2583-2594 Tanaka K, Yoshimura T, Tamura T, Fujiwara T, Kumatori A, Ichihara A (1990) Possible mechanism of nuclear translocation of proteasomes. FEBS Lett 271 41-46... 

T. reesei culture filtrate by gel chromatography (10), contained both the 20 kDa xylanase, another glycanase with high laminarinase and low xylanase activity, and a hitherto unidentified esterase. When supplemented with / -xylosidase, the xylanases and esterase of this preparation released about half of the xylose and acetic acid of the substrate (Table IV). When purified acetyl esterase was added, the yields of xylose and acetic acid increased further about 1.6-fold. 

Up to the beginning of the 1990 s most of the work on thiooligosaccharides was devoted to their synthesis. During the last few years, tailor-made complex or sophisticated thiooligosaccharides were obtained and used both in biochemical and X-ray studies for getting a better understanding of the mechanism of action of glycanases. 

Although most of the glycosides 6-12 showed some inhibition towards the j3-glycanases available to us (18], it was the epoxybutyl -cellobioside 7 (n = 2) that was an efficient inhibitor of one particular enzyme. Also, in view of the chiral nature of an enzyme, it seemed an especially good idea to prepare molecules such as 7 in optically-pure form. We therefore began a program to synthesize the pure diastereoisomers 6a,6b,7a and7b. 

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