Lewis Blood Group Fucolipids and Their Isomers from Human and Canine Intestine*

Glycolipids containing fucose linked to N-acetylglu- cosamine were isolated and characterized from 14 individual human and 13 individual dog intestines. From 8 of the dog intestines, Lewis a isomer fucolipids were isolated, all identical and having the structure Gal(P1 --f 4)Fucal --f 3]GlcNAc@l + 3)Gal(Pl + 4)Glc-cer- amide. Lewis b isomer fucolipids were isolated from 12 of the intestines, all identical and having the structure Fuc(a1 += 2)Gal(P1 + 4)[Fucal "-f 3]GlcNAc(Pl + 3)Gal(P1 4 4)Glc-ceramide. Lewis a-active glycolipids were isolated as the sole major fucolipid in 6 of the human intestines and differed from the canine isomer only in the position of the linkage of galactose to N- acetylglucosamine, having the p1 + 3 (type 1) rather than the pl- 4 (type 2) linkage. Lewis b-active fucoli- pids were isolated from 8 human intestines and differed from their canine isomer only in that they, too, had the type 1 rather than the type 2 oligosaccharide

Glycolipids containing fucose linked to N-acetylglucosamine were isolated and characterized from 14 individual human and 13 individual dog intestines. From 8 of the dog intestines, Lewis a isomer fucolipids were isolated, all identical and having the structure Gal(P1 --f 4)Fucal --f 3]GlcNAc@l + 3)Gal(Pl + 4)Glc-ceramide. Lewis b isomer fucolipids were isolated from 12 of the intestines, all identical and having the structure Fuc(a1 + = 2)Gal(P1 + 4)[Fucal "-f 3]GlcNAc(Pl + 3)Gal(P1 4 4)Glc-ceramide. Lewis a-active glycolipids were isolated as the sole major fucolipid in 6 of the human intestines and differed from the canine isomer only in the position of the linkage of galactose to Nacetylglucosamine, having the p1 + 3 (type 1) rather than the pl-4 (type 2) linkage. Lewis b-active fucolipids were isolated from 8 human intestines and differed from their canine isomer only in that they, too, had the type 1 rather than the type 2 oligosaccharide chain.
Lewis a and b glycolipid isomers commonly co-existed in canine intestine as major fucolipids whereas Lewis a and b glycolipids did not so co-exist in human intestine. In all of the fucolipids, only hydroxylated fatty acids were present and phytosphingosine and sphingosine were the predominant long chain bases. These findings are of interest in the biosynthesis of these substances and in their genetic expression.
Human and canine intestinal epithelia contain a variety of fucolipids having AB0 and Lewis blood group activity. Most of these glycolipids are ceramides of simple oligosaccharides of 5 to 7 sugars, having the antigentically specific terminal carbohydrate structure. The structure and properties of these substances isolated from a variety of tissues have been re-* This work was partly supported by National Institutes of Health Grant CA-13148 to the University of Alabama; Birmingham, for procurement of tissues. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
.+ To whom correspondence should be addressed. Foundations.
viewed (1-3). Of immunological interest is the group of fucolipids in which fucose is linked to N-acetylglucosamine, a characteristic of blood group glycoproteins and glycolipids with Lewis blood group activity. Some of the glycolipids have Lewis a and Lewis b antigenic activity and others have little or no activity (4-6). This paper reports data on the characterization of 2 simple types of these intestinal glycolipids. Also reported are data on the distribution of these and other fucolipids isolated from 37 individual dog small intestines and 16 individual human small intestines.

EXPERIMENTAL PROCEDURES'
EXPERIMENTAL ELETHODS I. Isolatron Of the FUCOIIpidS ter experimental surgery. The human small lntestlnes were all qzossly normal and taken at autopsy, usually wlthrn 4 01 5 hours Post morrem. The tlssues were extracted with ethano1:ether 0 : 1 1 as dercrlbed ore-Normal dog Small intestines were taken lmediately at SaCIlfIce afviausly I11 except that 10 m l Of Solvent mlxture were Used per gram Of fresh tissues for the inltlal extractmn. and 3 ml/q for the Second ext r d c r m n .
The fucolipids were lsolated from these extracts by s111clc acid and FIorls11 chromatography, solvent partltlon. and preparative thin layer chromatography as described prev~ously 1 8 ) . Before derlvatlzatlon for ma95 spectrometry and NMR SpeCtroSCOPy the LewlS a 1SOmeT (dog 3 8 F-1) and the Lev15 b isomer (dog 38 F-2 and 4 4 F-2 Put toqetherl were acetylated ~n acetlc anhydride. w r i d m e and chloroform 1:1:1 Iv/v/v) ocer n q h t at roam tenperaturi-and sublected to preparative thln layer chromatography wlth chloroform and methanol 96:4 ("/VI. Band3 VIsualized wlth m d u e vapours were scraped Off and put on a s11lc1c acld column. The acetylated glycolipid was eluted wlth chloroform and methanol 2:1 ( " / V I and deacetylated I" 0.1 ml Of toluene, 0.1 ml Of methanol C o l m Of 0.1 g Of Amberllte (CG 5 type I in Hi form) and after eluted and 0.2 ml of 0.2 H KOH 1" methanol. The mixture was passed through a Wlth methanal.

Determination Of the Constituent Sugars
The fucolipids were hydrolyzed, the liberated suqars reduced to the et a1 1 9 1 . The aldltol acetates were then analyzed by gas chromatography aldztolr and acetylated by the methods Of Yang and nakomorl 161 and Smith, I S described preYIous1y 191. The procedures for Oxldation of the intact llplds with Sodlum permdate and for the determmatmn of s u q a r~ r e m a nzng after oxidatmn have been descrrbed (9).

E .
Selective Hydrolysis of the FucoIqldS selective removal of the Fucose residues for enzyme degradatum and methylation studies was carrred out as follows. Hellm gas co1-flow was 45 ml/min. This p r o g r m was used for resolution of the 3.6 and 4.6 di-0-methyl derivatives Of N-acetylglucosamine from glycolipids with fucose removed.

D. Enzymes Degradatron Enzyme degradation Of the fucolxpid or ita defucaeylated der1VatlVe
was carried out with Jack bean E-N-a~etylhexosaminld~~=, Jack bean 8galactosidase and fig o-galactosidase a8 described prevloualy (91. Degradation of the fucolipids with an endo-8-galactosldase from Escherichia freundii bas also been described (111.

E. Analysis Of the Ceramides
The distribution of the Comwnent fatty acids and long chain bases of the dog intestine Lewis a lsbmer and Leiis b isomer fucoliplde was determined by gas liquid chromatography of the methyl eaters or the trimethylsilyl derivatives and aldehyde derivatives respectively as deecribed previously (121. The composition of the ceramzdes Of the Lewis a and the Lewis b lsomer fucolipids has also been determined by mass spectrometry (13.141. The comwasition Of the Lewis b fuwlxpid ceramldes determined by thls method-1s given below.

Dog
Intestinal Lewis a Isomer Fucolipid-This substance was isolated from 8 of 37 dog intestines as a major fucolipid.
In 1 intestine, it was the sole major fucolipid; in the other 7, the Leb isomer fucolipid was also present and in 4 of the latter, the difucosyl A glycolipid was present as well ( Table I).
Results of sugar analysis of the Le" isomer fucolipids indicated that they were pentaglycosyl ceramides containing glucose, galactose, N-acetylglucosamine, and fucose in a molar ratio of 1:2:1:1, respectively. Of these, only galactose, glucosamine, and small amounts of glucose remained after oxidation of the intact lipids with periodate. The lipids are isomers of both the dog blood group H and human blood group Le" fucolipids but do not have these immunological activities (5).
Five of the 8 isolates have been permethylated and 3 of these have also been permethylated after selective removal of fucose. Typical gas-liquid chromatograms of the partially methylated alditol acetates resulting from these 2 methylations of the fucolipid of dog 51 are given in Fig. 1A. The 3,6di-0-methyl-N-acetylglucosamine derivative has replaced the 6-0-methyl-N-acetylglucosamine present in the products of the intact fucolipid. Fucose and galactose are therefore linked at the 3 and 4 positions of the N-acetylglucosamine, respectively.
The glycolipid was not hydrolyzed by an a fucosidase from Turbo cornutus (Miles Laboratories) and from bovine epididymus (Sigma) but was hydrolyzed to a glucosyl ceramide by the endo-@-galactosidase from Escherichia freundii.
Analysis of the ceramide fatty acids and long chain bases from the fucolipid of dog 31 is given in Table 11. All of the fatty acids were hydroxylated and there was a fairly even distribution of even numbered carbon fatty acids from C N to CZ4. Phytosphingosine was the predominant long chain base.
The Ouchterlony double diffusion-in-gel precipitin test is shown in Fig. 2. The defucosylated glycolipid of dog 31 formed smooth precipitation bands which fused into symmetrical arcs with those of neolactotetraosyl-ceramide, indicatlng the identity of the 2 haptens.  Mass spectra were recorded of the permethylated derivative ( Fig. 3) and of the permethylated-reduced derivative (not reproduced). The reduced derivative produced a spectrum which was very similar to that of the same derivative of the Lewis a glycolipid published before (13). Thus, a series of peaks at m/e 1359, 1377, 1415, 1443, 1457, and 1471 corresponded to the complete saccharide (1 fucose, 1 hexosamine, and 3 hexoses) and a varying hydroxy fatty acid with 16, 18, 20, 22, 23, and 24 carbon atoms, respectively. A peak at m/e 1403 was evidence for a 2 position of the fatty acid hydroxyl and that phytosphingosine was a major base. The sequence of the 5 sugars was possible to deduce from the spectrum of the nonreduced derivative (Fig. 3). Terminal fucose (m/e 157 and 189), terminal hexose (m/e 187 and 219) and the hexosamine together produced the trisaccharide ions indicated at m/e 606 and 638 (see top of Fig. 3). These data and the tetrasaccharide peaks at m/e 810 and 842 exclude alternate sequences to that given by the formula. The ceramide peaks at m/e 706 and 722 support the conclusion that the heaviest species present in significant amounts contained phytosphingosine and 2-hy-&oxy 24 carbon fatty acid.
NMR spectra were recorded for both permethylated and permethylated-reduced derivative. Part of the latter is reproduced in Fig. 4, spectrum 1. The 5 anomeric protons indicated were concluded from comparison of spectra of similar glycolipids with or without fucose (19-21). The @-glucose doublet is located most high-field followed by the 2 ,&galactose signals, the P-glucosamine signal, and far down-field the a-fucose at I  I  I  I  I  I  I  I   5.80 ppm (J1,2 = 2.7 Hz). Before reduction, the a-fucose signal was found at 4.79 ppm ( J I ,~ = 3.9 Hz). This great change in chemical shift upon reduction is probably due to a deshielding effect of nitrogen and is found for anomeric protons of sugars in position 3 of the glucosamine (17,20,21). Therefore, the down-field a-signal (small coupling constant) should be due to fucose in position 3 of glucosamine. The resonance shown close to the glucosamine, signal was probably caused by a nonanomeric proton, possibly H-5 of fucose.
This is a ceramide of lacto-N-fucopentaose I11 and identical with that isolated by Yang and Hakomori (6) from human adenocarcinoma tissue and which they have termed "human tumor glycolipid." Dog Intestinal Lewis b Isomer Fucolipid-This glycolipid is separated from the blood groups H, A, and Lewis a isomer fucolipids by its lower mobility in preparative thin layer chromatography with silica gel. It has been isolated from 12 of the 37 dog intestines as a major fucolipid: in 2 intestines as the sole major fucolipid, in 2 along with blood group H fucolipid, in 3 with the Lewis a isomer fucolipid, in 1 with the difucosyl A lipid (13), and in 4 with both Lewis a isomer and difucosyl A lipids (Table I).
Results of the sugar analysis of the Lewis b isomer fucolipids clearly show that they are hexaglycosylceramides containing glucose, galactose, N-acetylglucosamine, and fucose in a molar ratio of 1:2:1:2, respectively. Treatment of the fucolipids of 3 of the dogs (numbers 13, 16, and 23) with periodate resulted uniformly in decomposition of all sugars except 1 mol each of glucosamine and galactose. They are isomers of the human blood group Lewis b fucolipids and share at least some of the latter's immunological activity (5).
Several of the isolates have been permethylated including some with small amounts of the difucosyl A glycolipid contaminant. Gas-liquid chromatograms of the partially methylated alditol acetates resulting from methylation and methylation after defucosylation are given in Fig. 1B   Enzyme degradation of the defucosylated glycolipid of dog 16 F2 is shown in Fig. 5. Jack bean P-galactosidase converted the lipid to a product migrating with a standard trihexosyl ceramide; the p galactosidase and P-N-acetylhexosaminidase together converted it to an apparent monohexosyl ceramide. Incubation with ,&galactosidase, then heating to inactive, and incubation of the product with the P-N-acetylhexosaminidase gave a product which migrated with a dihexosyl ceramide. The defucosylated glycolipid was not hydrolyzed by fig a-galactosidase. These results indicate a Galp + GlcNAcP + Galp + Glcceramide structure.
Analysis of the ceramide fatty acids and the long chain bases from the fucolipid of dog 31 is given in Table 11. All of the fatty acids were hydroxylated and only 25% were below 20 carbons in chain length. Sphingosine (58%) and phytosphingosine (38%) accounted for nearly all of the long chain bases. Mass spectra of the Leh isomer fucolipid of dog 16 have been published (14) and clearly demonstrate the structure: fucose-hexose-[fucose]-hexosamine-hexose-hexose-ceramide. These spectra also c o n f m t h e composition of the ceramide fatty acids and long chain bases given in Table I of the Leh isomer fucolipid of dog 31. Only 2-hydroxy fatty acids were found and phytosphingosine was a major long chain base.
Rabbit antiserum directed against dog 23 Lewis b isomer fucolipid reacted on Oucherlony plates with solutions of this fucolipid and with Lewis b isomer fucolipids from dogs 9, 13, 16, and 31 giving a continuous band around the center well (Fig. 6). This is evidence for the identity of these 5 fucolipids.
NMR spectra were recorded for both permethylated derivative (not reproduced) and permethylated-reduced derivative of the Lewis b isomer prepared from dogs 38 and 44 and put together (Fig. 4, spectrum 2 ) . In agreement with the enzyme degradation, 2 /3-galactoses and 1 P-glucosamine were concluded (19)(20)(21). In addition, a ,&glucose signal was found for both derivatives. Two a-resonances typical for the 2 expected fucoses are shown. The fucose in position 2 did change its chemical shift only slightly upon reduction. However, the fucose in position 3 of the glucosamine shown at 5.76 ppm ( J l . 2 less than 2 Hz) for the reduced derivative (Fig. 4, spectrum 2) had changed from a split signal a t 4.77 ppm (J1.2 = 3.2 Hz) and 4.83 ppm (JI, = 3.0 Hz) before reduction (not reproduced). This is conclusive for this position (21).
The above data are consistent with the following structure for the Lewis b isomer foculipid of dog intestine: Fuc(a1 Human Intestinal Lewis a Fucolipid-The structure of this fucolipid has been determined (13) on the basis of all of the above criteria except for methylation studies on the defucosylated lipid and NMR spectra. The fucolipid has been isolated from 6 of 16 human small intestines as the only major fucolipid present (Table I). The structure proposed (13), Gal(P1 + 3)[Fuc(al + 4)]GlcNAc(Pl + 3)Gal(/31 + 4)Glcceramide, has been confirmed by methylation studies on these fucolipids ( Fig. 1 0 and by the NMR spectrum of the permethylated-reduced derivative (Fig. 4, spectrum 3). Compared with the nonreduced derivative (spectrum not shown), a , f 3resonance has moved down-field which is additional evidence for a Gal(P1 + 3)GlcNAc linkage (20,21). The P-glucose signal had the typical high-field position (19)(20)(21).
Human Intestinal Lewis b Fucolipid-This glycolipid was separated from the blood groups H, A, and Lewis a fucolipids by its lower mobility in preparative thin layer silica gel plates, as was the case with the dog intestinal Lewis b isomer fucolipid. It has been isolated from 8 of 19 human intestines as a major fucolipid: in 5 of these as the only major fucolipid, in 2 along with the difucosyl A glycolipid, and in 1 with an uncharacterized high molecular weight fucolipid. All of the lipids of this group were hexaglycosylceramides containing glucose, galactose, N-acetylglucosamine, and fucose in a molar ratio of 1:2:1:2 and by these criteria were identical with the dog intestinal Lewis b isomer fucolipid. Mass and NMR spectra of the permethylated and permethylated-reduced derivatives,

Lewis Blood Group Fucolipids and Their Isomers
enzyme degradation studies, and permethylation studies before and after defucosylation all gave data entirely consistent with that of a ceramide of lacto-N-difucohexaose I as proposed in a preliminary note by Hakomori and Andrews ( 4 ) for the Lewis b-active glycolipid they isolated from human adenocarcinoma tissue.

DISCUSSION
Methylation analyses of 10 dog intestinal mono-and difu-cosy1 glycolipids having fucose linked to N-acetylglucosamine, 6 fucolipids with blood group H, and 9 with blood group A specificities have shown that galactose is linked exclusively to position 4 of N-acetylglucosamine. In contrast, analyses of 10 human Lewis intestinal fucolipids, 4 difucosyl A glycolipids, and 1 with blood group H specificity have shown the galactose linked only to position 3 of N-acetylglucosamine. The NMR spectra of reduced derivatives show characteristic and great differences in chemical shifts for a-fucose and P-galactose, whether in position 3 or position 4 of glucosamine (Fig. 4).
The distribution of the several types of fucolipids isolated from 37 individual dog intestines and 16 individual human intestines is given in Table I. These were all major fucolipids in the sense that 6 to 40 mg were recovered from the dog and 8 to 80 mg from the human small intestine. Most of the intestines contained other fucolipids in very small quantities, usually in unresolved mixtures comprising 2-5 mg of material. There were 2 major unresolved mixtures and 1 complex major fucolipid that has not been characterized. Under these circumstances there are limitations in interpretation of these data in genetic or immunologic terms.
In dog intestines, the most common fucolipids were the blood group A and Lewis b isomer present in about a half and a third of the samples, respectively, whereas the Lewis a and Lewis b predominanted in 14 of the 16 human intestines. The latter did not occur together in these adult human intestines as has been reported in human cecal tumors (4). Blood groups A and H fucolipids appear to be less common in human than in canine intestines.
The findings in both dog and human intestine show marked differences in genetic expression between small intestine and red blood cells. In the dog, the AB0 and Lewis isomer fucolipids are prominent in intestine but are not present in blood and are not related to canine blood typing. In man, the most common intestinal fucolipids, Le" and Leb, do not appear in red blood cells until transferred there from the plasma proteins (22). The most common human erythrocyte fucolipids, A and H, are less common in intestine and are expressed there with the type 1 chain, whereas these are nearly all of the type 2 chain in human erythrocytes.
Although no Lewis isomer fucolipids were found in human intestine, their precursor, neolactotetraosyl ceramide, is available to bone marrow, and in in vitro experiments, a fucosyl transferase in human blood plasma converted it to Lewis a isomer fucolipid (23). However, these isomers were not identified in a systematic isolation and resolution of human plasma glycolipids in which Lewis a and Lewis b fucolipids were isolated and identified (24,25). Lewis a isomer has been found in human adenocarcinoma tissue (6) and in normal and Krabbe diseased human brain (26,27).