Release of oligosaccharides from various glycosphingolipids by endo-beta-galactosidase.

The hydrolyzability of various glycosphingolipids catalyzed by endo-&galactosidase of Escherichia freundii has been studied. The lacto-N-glycosylceramide series having the common structure R -+ GlcNAc/31 + 3GalPl + 4Glc (or GlcNAc) were hydrolyzed at Gal/U + 4Glc (or GlcNAc) linkages. fl-Galactosyl linkages in “glob0 series” or “ganglio series” of glycolipids were not hydrolyzed. Oligosaccharides liberated from glycolipids were isolated, and their structures were identified by mass spectrometry. The kinetics of hydrolysis of various substituents of paragloboside and of Hzand HQ-glycolipids were studied. The following findings were of particular interest: 1) sialosyl substitution at the terminal galactosyl residue of lacto-N-neotetraosyl structure of paragloboside greatly enhances the hydrolyzability of the internal Gal/31 -+ 4Glc linkage, thus producing a high yield of sialosyltetrasaccharide (AcNeua2 + 3Galpl+ 4GlcNAcP + 3Gal); 2) the Gal/l1 + 4GlcNAc linkage locating in the middle of the repeating Gal/l1 + 4GlcNAc (or Glc) unit seen in Hzor Ab-glycolipid, (R + Galbl -+ 4GlcNAcPl + 3GuZfll + 4GlcNAcfil+ 3Galfil+ 4Glc + Cer) was preferentially hydrolyzed relative to the Gal/31 + 4Glc linkage directly attached to ceramide, and 3) the branched structure as is found in HZ-glycolipid greatly reduced the hydrolyzability. The H8-glycolipid was hydrolyzed at a higher concentration of enzyme, resulting in the liberation of a branched nonasaccharide which was, in turn, degraded into a branched heptasaccharide and a disaccharide.

Oligosaccharides liberated from glycolipids were isolated, and their structures were identified by mass spectrometry.
The kinetics of hydrolysis of various substituents of paragloboside and of Hz-and HQ-glycolipids were studied.
The H8-glycolipid was hydrolyzed at a higher concentration of enzyme, resulting in the liberation of a branched nonasaccharide which was, in turn, degraded into a branched heptasaccharide and a disaccharide.
Endo-/3-N-acetylglucosaminidases which specifically hydrolyze the internal glycosidic linkage in glycoproteins are becoming powerful tools for determining the structure and function of the carbohydrate moiety of glycoproteins (l-8). However, an endoglycosidase that can hydrolyze the carbohydrate moiety of glycolipids has not been described yet.
We have shown that endo-jl-galactosidase from Escherichia freundii can hydrolyze a glycoprotein from ovarian cyst mucin and oligosaccharides from human milk (9, 10); although this enzyme was originally found as a keratan sulfate-degrading enzyme (11,12 substrate susceptibility.
In addition, the specificity of the enzyme has been determined by structural characterization of hydrolysis products.
Oligosaccharides were isolated from the aqueous layer after partition by chloroform/methanol/water, reduced by NaB"H4 and applied to paper chromatography.
Each oligosaccharide alcohol showed a single peak corresponding to the position of oligosaccharide which shows the reasonable RF value as expected from the structure of the starting glycolipid. No monosaccharide or intermediate peak was observed (Fig. 3, A to F).
To identify the products further, oligosaccharides isolated from enzymatic hydrolysate of lacto-N-triosylceramide and sialosylparagloboside were reduced, permethylated, purified on thin layer chromatography and analyzed by mass spec- Glycolipid Hydrolysis by Endo-P-galactosidase 6815 trometry (Fig. 4, A and B). The mass spectrum of each 1" (see "Materials and Methods"), lacto-N-triosylceramide permethylated oligosaccharide alcohol indicates the structure and the major slow migrating oligosaccharide were produced shown in Fig. 4.
(R: H, Galbl -+ 4, Gala1 -+ 3Gal,% + 4, Galpl -+ 3Gal/31 However, with a higher concentration of enzyme ("Condi--+ 4, L-Fucol -+ 2Gal/?l-+ 4, and AcNeua2 + 3Gal/31-+ 4). tion 2"), Ha-glycolipid and Ah-glycolipid were hydrolyzed to The relative effect of the substituent group on the hydrolyz-Cer(Hex) and two oligosaccharides, respectively (see Fig. 5 Hydrolysis of Hi-Glycolipid--In contrast to HP-glycolipi H.S-glycolipid was hydrolyzed to Cer(Hex) and oligosaccharic only under "Condition 2" (see "Materials and Methods" Using a lower concentration of enzyme (such as under "Cor dition l"), hydrolysis of H.,-glycolipid was scarcely observe1 Thin layer chromatography (Fig. 6) and paper chromatol raphy of released oligosaccharides (Fig. 3iY)  to a heptasaccharide and a disaccharide lipids-Release of oligosaccharide from various glycosphin-(GlcNAcj31 + 3Gal) were released later as well. No lacto-2l'-oglipids was measured and shown in Fig. 7. The paragloboside triosylceramide was detected at any time. In addition, the group ( Fig. 7A) with sialosyl substitution at the terminal amount of nonasacharide released from H:l-glycolipid appear galactosyl residue greatly enhanced the enzyme susceptibility. to be maximum after 4 h and then decrease without reaching Other substitutions to the paragloboside structure slightly a 100% hydrolysis (Fig. 7C). Furthermore, the amount of affected the hydrolysis rate. Since the release of Fucal --, released heptasaccharide was almost equal to that of disac-2Galbl--+ 4GlcNAcPl-+ 3Gal from HZ-glycolipid (Fig. 7B) is charide during hydrolysis. much faster than that from H,-glycolipid ( Fig. 7A), the en- The structure of nonasaccharide was confined by mass zyme can hydrolyze the P-Gall + 4 bond in Hz-glycolipid spectromery after reduction and permethylation (Fig. 40). which is locating at the distance of 4 sugar residues from the The minor oligosaccharide component, which migrates ceramide moiety than that which is linked directly to the Glc slightly faster than nonasaccharide on thin layer chromatog--+ Cer moiety. The release of GlcNAcj31 + 3Gal from HPraphy after methylation, showed m/e 189, 393, 638, and 204. glycolipid (Fig. 7B) was slower than that from lacto-N-trio- The results strongly suggest the presence of heptasaccharide sylceramide (Fig. 7A), and appears to have a lag time. This since branched reducing terminal galactitol gives m/e 204. fact suggests that the release of GlcNAcPl + 3Gal from Hz-These results indicate that HZ,-glycolipid is hydrolyzed essentially as follows: Glycolipid Hydrolysis by Endo-/3-galactosidase 6817 glycolipid does not occur until Fuccrl + 2Gal/?1 + 4Gaw1 --, 3Gal is liberated. H.7-glycolipid was hydrolyzed very slowly (Fig. 7C), possibly due to the presence of a branched galactose residue which is hardly hydrolyzed and in some manner restricts the enzyme access to the more internal galactosidic bond.
In this study, we found that this enzyme can hydrolyze Aand H-active glycolipids. However, the intact AB-active glycoprotein was not hydrolyzed previously (10). This discrepancy should be studied further. Because a high concentration of enzyme was necessary to hydrolyze Ha-glycolipid, it is possible that the AB blood group glycoprotein could be hydrolyzed with a large amount of enzyme.
Endo+galactosidase is capable of hydrolyzing various substrates such as keratan sulfate, glycoproteins from mu&s, and oligosaccharides from human milk (9, 10). It is now apparent that the enzyme is also capable of hydrolyzing various glycosphingolipids of the "lacto series." Since this class of glycolipid is the carrier of antigenic determinants of blood group ABH, Lewis (17,18,34), Ii (17,35,36), PI (37), and p (38) and probably for tumor-associated antigens (24,39,40), the enzyme will be useful to analyze and modify the antigenicity of membrane antigens in cellular extracts and on cell surfaces in situ. It is particularly noteworthy that the enzyme is capable of hydrolyzing the internal /?-galactosyl linkage without removal of nonreducing sialosyl termini; thus, direct modification of cell surface antigens is possible by applying the enzyme on cell surfaces. An obvious change of ,' According to the nomenclature recommended by IUPAC-IUB Commission on Biochemical Nomenclature, The Nomenclature of Lipids; (1977) Lipids 12,455-468. the antigenicity of human erythrocytes has been observed by treating erythrocytes with this enzyme (41). The enzyme has therefore great potential as a tool for structural analysis and functional modification of cell surface glycoconjugates.