THEJOURNALBIOLOGICAL OF CHEMISTRY

Vol. 259,No.11, Issue of June 10,pp. 7178-7186,1984 Printed i U.S.A. n

Immunochemical Studies on Blood Groups

PURIFICATION AND CHARACTERIZATION OF RADIOACTIVE 3H-REDUCED DI-TO HEXASACCHARIDES PRODUCED BY ALKALINE /3-ELIMINATION-BOROHYDRIDE REDUCTION OF SMITH DEGRADED 3H BLOOD GROUP A ACTIVE GLYCOPROTEINS*
(Received for publication, October 31,1983)

Albert M. WuS, Elvin A. KabatQTJI, Nilsson**$$, David A. Zopf**, Flavio G. GruezoQ,and Bo Jerry LiaoQ
From the $Department of Veterinary Pathlogy, College of Veterinary Medicine, Texas A&M University, College Station, Texas 77843, the Departments of Microbwlogy, Human Genetics and Development, and Neurology, and the Cancer Centerllnstitute for Cancer Research, Columbia University, College of Physicians and Surgeons, New York,New York 10032, the lINational Institute of Allergy and Infectious Diseases. and the **Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Betheida, Maryland 20205

Treatment of bloodgroup A active glycoprotein from horse B substances contained various a-linked L-fucohuman ovarian cyst fluid by one stage of Smith deg- pyranose and a-linked Gal substitutions on the comradation followed by alkaline &elimination in the pres-posite structure. A, B, and H-active oligosaccharides, ence of NaB[H4] (Carlson degradation) liberated tri- formed after alkaline borohydride treatmentof blood tiated oligosaccharide alditols. The carbohydratemix- group substances under conditions which resulted in ture was fractionated by gel filtration, elution from alkaline 8-elimination followed by peeling, likewise charcoal, paper chromatography, and high pressure contained a-linked GalNAc, Gal, and L-fucopyranose liquid chromatography. Structures were established on similar core structures but with 3-hexenetetrols, based on sugar composition, periodate oxidation, meth- hexanepentols, andgalactitolatthe reduced ends. ylation analysis, and analysis oligosaccharide aldi- Present resultsconfirm the extensiveheterogeneity of of tols as permethylated and N-trifluoroacetylated deriv- blood group glycoproteins described previously. atives by gas-liquid chromatography-mass spectrom- Whether this is merely incomplete biosynthesisor is a etry.The following structureshave been deduced: consequence of programmed regulatory control glyby Gal/31+3GalNAc-o1, GlcNAc~1+6GalNAc-ol, G a u l + cosyltransferases deservesfurther study. 3GlcNAc~1+6(3-deoxy)GalNAc-ol, Gal/31+3GlcNAcBl+GGalNAc-ol, Gal@1+4GlcNAc/31+6GalNAc-ol, GlcNAc~1+3Gal@1+3GalNAc-ol, Galj31+3[GlcNAc@1+6]GalNAc-ol, G a l ~ l + 3 [ G a l ~ 1 + 4 G l c N A c ~ 1 + 6 ] - The internal structure of the carbohydrate moiety of the GalNAc-01, Gal~1+3GlcNAc~1+3Gal~l+3GalNAc- water-soluble human blood group active glycoproteins, puri01, GlcNAcB1+3Galj31 +4GlcNAcBl +GGalNAc-ol, fied from human ovarian cyst fluid, was not resolved until GlcNAc/3l+3Ga1/31+3[Ga1~1+4GlcNAc~l+6]Gal- base was applied to cleave the 0-glycosidic linkage between NAc-ol,Gal~1+3GlcNAc81+3GalB1+3GlcNAc~1+3- the GalNAc of the carbohydrate and Thr and Ser of the Galj3l~3GalNAc-ol,Ga~1+3GlcNAc~1+3[Ga431+4GlcNAc~1+6]Gal@1+3GalNA~-ol,Galfil+3GlcNAc- protein core (1-6). Previously proposed composite structures of the carbohydrate side chains of blood group A, B, H, Le ~l+3GalB1+3[Gal81+4GlcNAcj31+6]GalNAc-ol. The smaller structures represent pieces of the larger and Leb substances and of precursor substances with I and i structures. Together they provide directevidence for determinants (7-10) were inferred from the mechanism of the the core structure of the carbohydrate side chains in alkaline /3-elimination and peeling reactions, and the structhe blood group substancesas proposed by K. 0. Lloyd tures of isolated oligosaccharides (8).More evidence is needed Proc. Natl. Acad. Sei.U. S.A. to confirm this carbohydrate core structure. Alkaline /3-elimand E. A. Kabat ((1968) 61, 1470-1477). Oligosaccharides previously isolated ination of intact blood group substances liberates large after Carlson degradation intact human ovarian cyst amounts of nondialyzable oligosaccharides and glycopeptides. of human and Smith degradation of blood group substances priorto alkaline fluid HLeb, Le*, and B substances and from borohydride treatment decreases the size of the more highly substituted nondialyzable carbohydrate chains by removal of *This work was supported in part by Grant CA 13696 to the Cancer Center, Columbia University, and, since December 1981, in terminal blood-group active sugars whose structures are alpart by Cooperative Agreement 58-519B-0-880(Project H-6194) ready well known (2-7, 11-18). As a result, core oligosacchabetween the United States Department of Agriculture and the Texas rides from longer chains become available for structural analAgricultural Experiment Station, TexasA&M University; and by the ysis. Addition of [3H]-borohydride during the alkaline pUnited States Department of Agriculture Formula Animal Health elimination-borohydride reduction provides a radioactive Funds, Project 6648. This article is No. LXXI of a series; the previous article isRef. 21. The costs of publication of this article were defrayed marker to ensure that oligosaccharides are reduced and thus
~~

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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. 1) Recipient of Grants PCM 76-81029 and 81-02321 from the National Science Foundation. $4 Present address, University of Lund, Chemical Center, Department of CarbohydrateChemistry, Post Office Box 740, s-22007, Lund, Sweden.

The abbreviations used are: GalNAc, 2-acetamido-2-deoxy-~-galactopyranose; Gal, D-galactopyranose; Fuc, L-fucopyranose; GlcNAc, 2-acetamido-2-deoxy-~-glucopyranose; GalNAc-ol, 2-acetamido-2deoxy-D-galactitol; Gal-ol, galactitol; hexNTF, 2-deoxy-2-trifluoroacetamidohexosyl; hexNTF-ol, 2 -deoxy- - trifluoroacetamidohexitol; 2 glc-ms, gas-liquid chromatography-massspectrometry; Hplc, high pressure liquid chromatography.

7178

Blood Group Carbohydrate Structures

7179

represent the alditol forms of the original 0-linked chains N NaOH and 1 M NaB[3H,] at 50 C for 16 h (Carlson rather than products of a peeling reaction (19, 20). Experi- degradation) (22) liberates reduced oligosaccharides with 3H reduced end. As described previously (21), the liberated ments in which blood group determinants and/or sugar resi- at the dues at nonreducing ends of a human blood group A active reduced oligosaccharides were fractionated by dialysis, folsubstance were removed by sequential Smith degradations lowed by retardion, Bio-Gel P-2, P-4, and P-6 columns. They were describedpreviously (21). Changes of biological activities were further purified on charcoal-celite columns, and by preand carbohydrate composition were compared before and after parative paper chromatography and hplc. Among carbohySmith degradation. The oligosaccharides with 3H at the re- drate side chains isolated, 77% contained less than 6 sugars. duced end were prepared by alkaline @-elimination and si- These were further purified by hpic and identified by glc-ms multaneous radioactive [3H]borohydride reduction (Carlson as permethylated N-trifluoracetyl derivatives and/or by perdegradation) (22, cf. 23). The size distribution of the carbo- iodate oxidation. As shown in Fig. 2, 14 different reduced 3Hhydrate side chains was also estimated; about 77% of the oligosaccharides were obtained. There are three hexasaccharcarbohydrate side chain residues contained from 1to 6 sugars, ide isomers ( R I M S 1.17), 2A, 2B, and ZC; one pentasaccharide, 13% from 7 to perhaps12 sugars and 10% were nondialyzable 3C ( R I M 5 1.5); three tetrasaccharides, the isomers 5 RIM^ 2.2, (glycopeptide fragments and/or polysaccharides). Among the RLac0.85),4B (RIMB 1.73, R, 0.65), and 16C major; four carbohydrate side chains of less than 6 sugars which were trisaccharide isomers, 6 major ( R I M 5 2.6), 6 minor ( R I M 5 2.6), isolated, 10% appeared various reduced furane chromogens, 7 (RIMS as 2.81, and 8B ( R I M 5 3.1); and the tetrasaccharide 8A indicating that about 10% of the carbohydrate side chains (RIMS 3.1) and two disaccharides 10 (Rhc 1.21) and 9B (Rhc had been degraded by peeling (20). The size distribution of 1.1).Ten had not been reported. The structuresindicate that carbohydrate side chains containing from 1-4 sugar residues the smaller oligosaccharides probably represent elements of contributed 15-25 mol %, about 79% of the totalcarbohydrate the larger oligosaccharides and could be a consequence of side chain residues of this fraction. incomplete biosynthesis of carbohydrate side chains. In the present report, the oligosaccharides with 3H at the These structures, especially the three hexasaccharide isoreduced end were further purified by high pressure liquid mers 2A, 2B, and 2C (Fig. 2) provide direct evidence for the chromatography and identified by gas-liquid chromatography- composite core structure of the carbohydrate side chains in mass spectrometry. Fourteencarbohydrate structures were the blood group substances as proposed by Lloyd and Kabat isolated and identified from the first Smith degraded glyco- (8). As shown in Fig. 9a, which is slightly rearranged from protein. Ten have not previously been reported; seven be- previous versions in which Branches Iand I1 were at an angle, longed to threesets of isomers. These well characterized the composite accounts for all the oligosaccharides previously structures provide (a) direct evidence for the formulation of a isolated from human ovarian cyst blood group B (27), HLeb composite core structure of the carbohydrate side chains in and Le substances (7), and from horse B substances (26). the blood group substances as proposed by Lloyd and Kabat The structure of three branches (I, 111, and IV) in Fig. 9u is (8); (b) evidence for both a longer branch or a new immuno- the composite core polysaccharide of the first stage Smith dominant A and/or H determinant at Galpl4GlcNAc(lac- degradation as derived from the oligosaccharidesin this paper. tosamine) ends of Branch I11 (Fig. 9) and another possible The oligosaccharides previously isolated from HLeb, Le, and linkage Gal~l-+3GlcNAc@l+6GalNAc-ol this at branch; and B substances of various species which were not subjected to (c) solid evidence for a new branch Galpl4GlcNAc linked Smith degradation are listed in Fig. 9, b top. Ineach instance, @ 1 4 a Gal nearest to Ser or Thr of the protein moiety. only the oligosaccharides isolated which contained Fuc (HLeb, to The terminal nonreducing end of this new branch can be Le) or a-linked Gal(B) are given. Core structures of five substituted by either Fuc or GalNAc or both and probably is oligosaccharides not shown in Fig. 9 also fit branches 111 and the site of a new H and/or A determinant. IV of the composite structure regardless of the pattern of The composite structure permits one to locate all blood substitution of the fucose residues. The oligosaccharides degroup reduced oligosaccharides previously isolated by alkaline scribed previously (Fig. 9) fit perfectly into the composite core borohydride treatment of various blood group substances structure and show that incomplete biosynthesis may occur which had not been exposed to periodate.1It also accounts for extensively along branch I. Biosynthesis may continue with the presence of a-linked terminal GlcNAc previously recog- branching, can stop atbranches I11 and IV or continue along nized as an antigenic determinant in humansimmunized with branch I and Fuc substitutions may occur on anyof the three hog A + H substance (24) by the isolation of GlcNAcal-+3 branches. Thus, thereis evidence for four fucoses which could or 4 Gal-ol in hog blood group A + H substances treated with occur on any of the Gal and/or GlcNAc residues of branch 1. alkaline borohydride under peeling conditions (25) and subsequently by the isolation of GlcNAcal+4GkNAcpl-+ Moreover, branch IV can vary in length since it survived 3Galpl+3GalNAc-ol (EL 0.88) from horse B substance (26) Smith degradation and both branch I11 and branch IV might have and of Gal/3l-+3[GlcNAcal4Gal~l4GlcNAc~l4JGal- Fuc substitutions on either Gal or GlcNAc, or both, giving H or Le determinants. From the oligosaccharides NAc-ol (Tij RIM5 1.67) in human B substance after hydrolysis previously isolated under peeling conditions, branches I and in 0.05 M NaOH, 1 M NaBH4 (27). I1 may have Fuc substitutions on the Gal and GlcNAc or both and, in addition, GalNAc and Gal substitutions to give A and EXPERIMENTAL PROCEDURES AND RESULTS B determinants (32, 41). The A, B, and H oligosaccharides previously isolated with 1 4 linked hexenetetrols of branch DISCUSSION I1 were a consequence of alkaline peeling (32), in which the bracketed Treatment of one stage of Smith degraded blood group A 1,3- and 1,Clinked substitutionsof branch I and the active glycoprotein from human ovarian cyst fluid, with 0.05 residues were eliminated (32). Branch 111 in its substituted

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* Portions of this paper (including Experimental Procedures, Results, Tables I and 11, Figs. 1-8, and Footnote 3) are presented in miniprint at theend of this paper. The abbreviation used is hex, hexosyl. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal

of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 83M-3033, cite the authors, and include a check or money orderfor $9.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

7180

Blood Group CarbohydrateStructures

I1 111 Gal G 1 a @1,4 U1.4 GlcNAc GlcNAc -1.6 ffl1.6 Ga1e1i3G1cNAcs1+3Galel*361cNAc8li3Galeli3GalNAc or d1.6

Serine

b
Fuc k1,4

Gal -1.4 GlcNAC3*1aFuc re1.6 Fuc Fuc h 1h 2 . 4 .1

i
Lea a c t i v e Lewis RIM 0.78 HLeb a c t i v e

Gal8li3GlcNAc8l~3Gal8li3GlcNAcsli3Gal8li3GalNAc-ol

Galali3GlcNAcsli3Galeli3GalNAc-ol

JS R 1 ~ 51.041
8 active T i j RIM 1.38

Gal rB1.3 GlcNAc4claFuc 4 . 3 Gal rs1,4 G1cNAc 4 . 6 Galeli3GalNAc-01 Ga 1 ia1.3 GalPcl~Fuc r81,4 G1cNAc r81.6 GalslJGalNAc-01 G1 a k1.3 GalZelsFuc +61,4 Fuc GlcNAc +61,6 k1,Z Galali3Gaiali3GalNAc-ol Fuc a t ,2 l Galeli3GalNAc-ol

Lea a c t i v e L e w i s R 1 ~ 5 0.47

d
e

Fuc -1.2

Galdi3Gal8li3GlcNAc~li3Gal8li3GalNAc-ol
Gal 481.4 GlcNAc +e1,6 M1.6 GlcNAc tB1.4 Gal

Fuc 4.4

B active T i j R 1 ~ 50.90 Horse 4 R 1 ~ 51.18

Galeli3GlcNAcsli3Galel,36alNAc-ol

Lea a c t i v e 1.28 Lewis RIM ( w i t h o u t Fuc, Lewis R 1 ~ 5 1 . 9 5 )

Fuc -1.4

B active T i j R1n5 0.36 Horse 4 RIM^ 0.65

H active Horse B R 1.38 T i j RL 1.$2 T j j RL 1.31
B active Horse RL 0.65,

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Galeli3GlcNAcsli3Galeli3GalNAc-ol
M1.6 G1cNAc M1.4 Gal Lea a c t i v e Lewis RIM 3.5

GalPcl~Fuc ffl1.4 FUC GlcNAcJel~Fuc -1.2 ~ 1 . 6 H active Galal+3GalNAc-ol JS RIM^ 0.9Z2 Gal ffl1.4 GICNAC c1.2 tB1.6 Gal~li3Galeli3GalNAc-ol
FUC

m
n

Fuc
*l.P Galal+3Gal81+3GalNAc-o1
GlcNAc C1.4 Gal 181.4 GlcNAc r61.6 GalBli3GalNAc-01
0.66. 0.68

active Horse R 1 ~ 51.15

Tj R 1 ~ 51.67 i

GalsldGlcNAc8li3GalNAc-ol
G1cNAca14G1cNAc614Ga181i3Ga1NAc-01

Horse B RL 1.04 Horse B RL 0.88

FIG. 9. A composite structure for the core of the water-soluble human A, B, H, Le, Leb, I, and i blood group glycoproteins based on H-reduced oligosaccharides isolated after Smith degradation and alkaline &elimination-borohydride reduction. Relation of previously isolated reduced oligosaccharides fromhuman HLeb, Le and human and horse B substances to the core structure. Data on JS and Lewis from Ref. 7, on Horse B from Ref. 25, and on Human B Tij from Ref. 39. The composite (a) is not intended to indicate that all substitutions can occur on a single GalNAc-Ser/Thr chain. Footnote 1, the alternate form Fuc Fuc JS J,a1,2 la1,3 could not be excluded but Fucd-+2Gal@1~3GlcNAc@1~3GalNAc-ol Gal~l4GlcNAc@1+3Gal@l-+3GalNAc RIM^ 2.35a was also isolated. Footnote 2, reduced oligosaccharideswith the same core structure but with two Fuc, one on DGal and one on DG~cNAc branch 1 1 JS Rm5 0.91; with two Fuc, one on each Gal JS RM 1.84, with of 1, I 5 1.80, 1; 1, one Fuc on DG~cNAc, Lewis RM 1.30 and JS R I M ~ of Branch 1 1 and with one Fuc on DGal of Branch 1 1 I 5 JS RM 1.90, and with one Fuc on the DGal of ~Gal@l-+3DGalNAc-ol, I 5 horse RL0.69, were also isolated.

form had a Fuc and Gal substitution to give a B determinant. It seems likely therefore that the Smith degradation could also have removed nonreducing Fuc and GalNAc from branches I11 and IV. In the present study, one of the hexasaccharide isomers 2C, the pentasaccharide 5, the tetrasaccharide 16C major, and the trisaccharide 7 (structures inFig. 2), also provide evidence for a longer oligosaccharide chain or possibly a new immunodominant A determinant at the lactosamine [Gal/31-*4GlcNAc]end (BranchI11 in Fig. 9a). The existence of such longer oligosaccharide branches was further supported by the tetrasaccharide 16C major. A related oligosaccharide, (Lewis R I M 5 0.47, Fig. 9i) was also found in the N-1 Lewisa active glycoprotein (7). Two trisaccharides 8A, Gal/3lt3GlcNAc/3l-*GGalNAc-ol the peeling product and BB, Gal~l-+3GlcNAc~l+6(3-deoxy)GalNAc-ol also provide new information about another possible 1-3 linkage at Branch 111. Such related elements are also found in the N-1

Lewis active glycoprotein (Lewis RL 0.44) (7) identical to 4B in Fig. 2. Thus, thecarbohydrate chains of Branch I11 can be For the Gal at the GalP1-A or 3GlcNAc~l+6GalNAc-o1. nonreducing end of the short chain in Branch I11 (GalB1t 4GlcNAc~lt6GalNAc-o1), 7 and 8B, to have survived Smith degradation, 7 and 8B would have had to be substituted by GalNAcalt3 or by Galal-3. This would not have taken place unless there had previously been a Fuccrl+2 at the nonreducing Gal to permitaction of an A or B glycosyl transferase (11, 12, 42). Thus, the short chain of 7 and 8B probably defines the location of a new A or B and H determinant. Similarly, 16C major would have had to have been substituted by Galpl-3 to have survived. Hence, the structure longer Gal~l+3GlcNAc/3lt3Gal~l~ 4GlcNAc~l+6GalNAc-ol could also have been the site of a potential A or B and/or H determinant. Indeed, Lewis R I M 5 0.47 (Fig. 9 i ) was isolated from Le substance and confirms

Blood Group Carbohydrate Structures

TABLE 111 Amounts of various oligosaccharides isolated in milligrams from H,L , and precursor Ii compared with B, e Le4 ' those fromfirst stage of Smith degradation of blood group A substance
Structure Amount
used

7181

Compound

18LS Gal,914GalNAc"ol GalNAc-ol (10)"

GlcNAc 181,6 Gal@143GalNAc-d

(6 minor)

Gal 181.4 GlcNAc GalP14GalNAc"ol (4B)

GlcNAc 18L6

(9B)

MSS A First IO, Beach B P1 (41) Precursor Ii OG (9)

Le' N-1 (7) HLeb JS (7)

2 3

Horse non-B active (26) 59 . Horse B active (26) 9.8 5.2 7.5 Human Tij 10% 2X BI (27) 8.4 6.4 6.4 Human Tij phenol insoluble B (27) a Compound numbers in Fig. 2 are in parentheses. In an earlier study under peeling conditions (19),12 mg were isolated from 3.0 g of N-1. If a value is not given in the table, the compound was not found.
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4.6 2.6 1.9 1.3 1.3 1.7

17 35 1 9 39 2.9 17.1

10

32

20
10.8 21 44b 2.8

5.5

the existence of a structure which without the F u c a l 4 on the GlcNAc (11) could have served as a precursor of H, A, and B determinants. 7 and 8B could also have been substituted by GlcNAc. The new branched hexasaccharide, Galpl"r3GlcNAcpl+ 3[Gal~l+4GlcNAc/3l4]Gal~l-+3GalNAc-ol (sample 2B) provides definitive evidence for a new branch (Branch IV in Fig. 9a), G a l ~ 1 4 G l c N A c ~ linked to the Gal nearest to 14 the Ser or Thr of the protein moiety. This new branch has also been found in the carbohydrate side chains of the N-1 Lewis" active glycoprotein (three such oligosaccharides were isolated in Ref. 7, Lewis R I M 8 1.95 and Lewis RIM^ 1.28, Fig. 3.5, 9e, and Lewis RIMs Fig. sf). This branch can be substituted by either Fuc or GalNAc or both andprobably defines the location of a new H andfor A determinant. 1 The existence of Branch 1 1in two different lengths permits one to incorporate the structure shown in Fig. 9n (Tij R I M S 1.67) intothe composite core structure. Evidence for an antigenic determinant specific for nonreducing terminal alinked GlcNAc had been obtained (24) in humans injected with hog A + H substance and by the demonstration that GlcNAcoll~3 4) Gal-ol was a potent inhibitor. Tij R I M S (or 1.67 (27) with a-linked GlcNAc can nowbe fitted into the composite structure. Some of the linkages (Fig. 9, o and p ) may be specific for horse blood group substances. The removal of Fuc and GalNAc and other terminal residues by Smith degradation has provided material for a more detailed analysisof the core structure of the A, B, H, Le', Leb, and Ii blood group substances. That the internalcore is very heterogeneous suggests that its biosynthesis can vary enormously. Thus, the individual chains 0-glycosidically linked by GalNAc to Ser or Thr may differ in length and sugar composition, even though all are synthesized according to a set of controlling factorsthat result in a single common core. Factors that determine the order of synthesis or preferentia1 elongation of specific chains are not known. The heterogeneity may not be haphazard but instead be influenced by may structural and regulatory genes controlling sites of biosynthesis and actions of glycosyl transferases in a manner more extensive than one has hitherto imagined. A t the moment, it is clear only that the chainswhich have been completed are those containing A, B, H, Le", and Leb determinants at their nonreducing ends. Since there is good genetic evidence that

the glycosyl transferases act in a defined order on precursor type structures, the reactions which result in chain termination have thus farinvolved addition of Fuc, GalNAc, and Gal in &-linkages;the existence of an antigenic determinant (24) in three species of specific blood group substances involving aterminal nonreducing a-linked GlcNAcmay be another instance of an a-linked chain-terminatingdeterminant which is not now known to have blood group activity. Its role in these glycoproteins requires further study. The compounds isolated in Fig. 2 provide a rough estimate of the amounts of substituted compounds in Fig. 9 which might be expected, in the sense that the first Smith degradation would leave precursor-like receptors (43) for the action of the various glycosyltransferases, in effect partially reversing what had taken place in the original biosynthesis of the various blood group substances. Destruction of subterminal residues in the blood group Asubstance used for Smith degradation would have been minimal because the GalNAc at the nonreducing termini of the blood group A determinants are linked a1-3 protecting the subterminal Gal. This Gal would have been destroyed on any chains on which biosynthesis had not gone to completion. Core structures would also have been destroyed had the Smith degradation been carried out on other thana blood group A or B glycoprotein. From 2 g of the first Smith degradation of MSS, 32 mg of compound 4B were obtained. Accordingly, one would expect that substantial quantities of blood group oligosaccharides built upon 4B would have been isolated from other blood group substances. This is most strikingly seen in JS in which five compounds in Fig. 9g and Footnote 2, with various fucose substitutions on 4B totalling 96 mg, were obtained from 2.56 g of JS, HLeb substance (7). Similarly, 17 mg of compound 10, Fig. 2, were isolated from 2 g of the Smith degraded A substance as compared with 5.9 mg of B active and 17 mg of H active compound which were isolated from 1.2 g of B active and 1.9 g of non-B active horse blood group substances and about 10 mg from human B substance 'Tij. Compounds 9B (10 mg), 7 (43 mg), 4B (32 mg), and 9j (4.7 and 2 mg) (Fig. 2) could have served for the synthesis of 9i (14.5 mg)and 9g and the four compounds with fewer fucoses (96 mg) (Fig. 9,Footnote 2). The extent of incomplete biosynthesis of these core structures can be estimated by comparing the yields given in Table

7182

Blood Group Carbohydrate Structures

6. Lloyd, K. O., Kabat, E. A., and Rosenfield, R. E. (1966) Biochemistry 5, 1502-1507 7. Rovis, L., Anderson, B., Kabat, E. A., Gruezo, F., and Liao, J. (1973) Biochemistry 12,5340-5354 8. Lloyd, K. O., and Kabat, E. A. (1968) Proc. Nutl. Acad. Sci. U. S. A. 61, 1470-1477 9. Vicari, G., and Kabat, E. A. (1970) Biochemistry 9,3414-3421 10. Pereira, M. E. A., and Kabat, E. A. (1979) Crit. Rev. Immunol. 1, 33-78 11. Watkins, W. M. (1980) Adu. Hum. Genet. 10, 1-136 and 12. Kabat, E. A. (1976) Structural Concepts in Immunology 13. 14. 15. 16. 17.
18. 19.

1 1 of the four compounds obtained from the first Smith 1 degradation of MSS and from Beach BP1, the nondialyzable product of mild acid hydrolysis of an ovarian cyst blood group B substance (41). Since these are yields, the data are necessarily approximate and the weights used and actual amounts isolated are given as compared with their yields from various undegraded blood group substances. Blood group substances are listed according to the number of genes required to go from the precursor Ii OG through Le" N-1, HLeb, JS and horse non-B active and B active, and human B substance Tij 10% 2X and Tij phenol insoluble. The data show decreased quantities of Gal~l+3GalNAc-ol,compound 10, obtained in HLeb, horse B active, andTij phenol insoluble in which additional glycosyl transferases had acted, as compared with the precursor Ii OG and Le" substances. The data on compounds 6 minor and 4B are similar; data on 9B are fragmentary. Thus, it appears that action of additional genes is accompanied by reduction in the numbers of incompletely synthesized interior residues. Outerarm and core heterogeneity in the oligosaccharide moieties of rabbit IgG, hybridoma, and myeloma immunoglobulins have recently been studied in relation to the x-ray crystallographic structure (44-46). In these glycoproteins there are two interacting complex type oligosaccharide chains, each joined to a C H domain: which may serve to separate ~ the domains. These have now been found to differ in outerarm sequence, with one chain lacking a terminal galactose on its a(1,3) arm such that a nonreducing terminal GlcNAc of one oligosaccharide chain bridges to, and interactswith, the mannosy1 chitobiose core of the opposing oligosaccharide chain. Moreover, the rabbit IgG was also heterogeneous with respect to the trimanosyl chitobiose core structure with 62% having the structure
(4)

Immunochemistry, 2nd Ed., Holt, Rinehart and Winston, New York Watkins, W. M. (1972) in Glycoproteins (Gottschalk, A., ed) 2nd Ed., pp. 830-891, Elsevier, Amsterdam Schiffman, G., Kabat, E. A., and Leskowitz, S. (1962) J. Am. Chem. SOC. 84,73-77 Schiffman, G., Kabat, E.A., and Leskowitz, S. (1960) J. Am. Chem. SOC.82, 1122-1127 Kabat, E. A. (1956) Blood Group Substances: Their Chemistry and Immunochemistry, Academic Press, New York Watkins. W. M.. Yates. A. D.. and Greenwell. P. (1981) Biochem. ,~ SOC.Trans. 9,' 186-191 ' Kabat, E. A. (1982) Am. J.Clin. Pathol. 78. 281-292 Lloyd, K. O., Kabat, E. A., and Licerio, E. (1968) Biochemistry
I~ I

7.2976-2990 20. Anderson, B., Rovis, L.,and Kabat, E. A. (1972) Arch. Biophys. Biochem. 148, 304-314 21. Wu, A. M., Kabat, E. A., Pereira, M. E. A,, Gruezo, F. G., and Liao, J. (1982) Arch. Biochem. Biophys. 215, 390-404 22. Carbon, D. M.(1966) J. Biol. Chem. 241, 2984-2986 23. Herp, A., Wu, A. M., and Moschera, J. (1979) Mol. Cell. Biochem. 23,27-43 24. Moreno, C., and Kabat, E. A. (1969) J. Immunol. 102, 13631367 25. Lloyd, K. O., Kabat, E. A., and Beychok, S. (1969) J. Immunol. 102, 1354-1362 26. Newman, W., and Kabat, E. A. (1976) Arch. Biochem. Biophys. 172,535-550 27. Maisonrouge-McAuliffe, F., and Kabat, E. A. (1976) Arch. Biochem. Biophys. 176,90-113 28. Kabat, E. A. (1961) Kabat and Mayer's Experimental Immuno-

Downloaded from www.jbc.org by guest, on January 11, 2010

Man
(1) (2) J d 6 (3)

chemistry, 2nd Ed. Charles C Thomas, Springfield, IL

29. Belcher, R., Nutten, A. J., and Sambrook, C. M. (1954) Analyst 79,202-208 30. Reissig, J. L., Strominger, J. L., and Leloir, J. F. (1955) J. Biol. Chem. 217,959-966 31. Winder, R. J. (1955) Methods Biochem. Anal. 2,279-311 32. Lloyd, K. O., Kahat, E. A., Layug, E. J., and Gruezo, F. G. (1966) Biochemistry 5, 1489-1501 33. Trevelyan, W.E., Proctor, D. P., and Harrison, J. S. (1950) Nature (Lond.)166,444-445 34. Whistler, R. L., and Durso, D. F. (1950) J. Am. Chem. Soc. 72, 677-679 35. Scott, C. D. (1974) Science (Wash. D.C.) 186, 226-233 36. Nilsson, B., and Zopf, D. (1982) Methods Enzyrnot 83,46-58 37. Nilsson, B., and Svensson, S. (1978) Carbohydr. Res. 62, 377380 K., and Chizov, 0. S. (1966) Adu. Carbohydr. 38. Kochetkov, N. Chem. 21,39-93 39. Nilsson, B., and Zopf, D. (1983) Arch. Biochem. Biophys. 222, 628-648 40. Nilsson, B., DeLuca, S., Lohmander, A., and Hascall, V. C. (1982) J. Biol. Chem. 257, 10920-10927 41. Lundblad, A., Hammarstrom, S., Licerio, E., and Kabat, E. A. (1972) Arch. Biochem. Biophys. 148, 291-303 42. Ginsburg, V. (1972) Adu. Enzymol. 36, 131-149 43. Schenkel-Brunner, H., Kabat, E. A., and Liao, J. (1979) J. Biochem. 98,573-575 Trans. 11, 44. Sutton, B. J., and Phillips, D. C . (1983) Biochem. SOC. 130-132 45. Rademacher. T. W.. Homans. S. W., Fernandes, D. L.. Dwek, R. A., Mizuochi, T., Taniguchi; T., and Kobata, A: (1983) Biochem. SOC. Trans. 11, 132-134 46. Rademacher. T. W.. and Dwek. R. A. (1983) in Progress in

Man@l4GlcNAc@14GlcNAc-Asn
p1,3

Man
(5)

with 15% having a F u c a l 4 linked either to GlcNAc (1) or (2); 13% having a GlcNAc linked 01-4 to Man (3); and 10% having both the F u c a l 4 and the GlcNAcal-4. Thus, heterogeneity is not restricted to the blood group glycoproteins and may be important tofunction. Analogous heterogeneities have been seen in otherN-glycans (for a review, see Ref. 47).
Acknowledgments-We wish to thank Dr. Garry Adams for his helpful comments on reading this manuscript and Linda Sroufe for her skillful typing. REFERENCES 1. Anderson, B., Hoffman, P., and Meyer, K. (1963) Biochim. Biophys. Acta 74, 309-311 2. Morgan, W. T. J. (1960) Proc. R. SOC. Lond. B Biol. Sci. 151,
308-347 3. Schiffman, G., Kabat, E. A., and Thompson, W. (1964) Biochemistry 3, 113-120 4. Rege, V. P., Painter, T. J., Watkins, W. M., and Morgan, W. T J. (1964) Nature (Lond.) 203,360-363 5. Kabat, E, A., Bassett, E. W., Pryzwansky, K., Lloyd, K. O., Kaplan, M. E., and Layug, E. J. (1965) Biochemistry 4, 16321638

' There are four domains in the heavy chain of IgG, one variable region VH and threeconstant region domains, CHI, c ~ 2 , cH3. and

Immunology V, Proceedings of the Fifth International 6ongress of Immunology, Tokyo, Japan, August 21-27,pp. 95-112, Academic Press, New York 47. Hatton, M. W. C., Marz, L., and Regoeczi, E. (1983) Trends Biochem. Sci. 8, 287-291

Blood Group Carbohydrate Structures

IWUWKHEMICAL STUDIES OM BLOW GRMPS. P U R l F l C A T l O l AN0 CHAQACTLRIlATlON O RAOIOACTIVE F [ 3 H ] REO LO OIL TO HEXA-SACCHAQIOES P R O W E O BY ALKALINE ~ - E L l ~ I N A l l O b 8 M I M Y D R l O f p H ] REOLTTION Of SMITH DEGRAMO B L W D GROW AACTIVE GLYCOPROTEINS A l b e r t M.

7183

Uu. E l v m A. Kabat. Bo f l i l r r o n .D a v i d
Flbvio6.

A. ZOpf,

GrueZo. a n d J e r r y L i a D

f X V f R l I E N l A L VRLXEOURES

Downloaded from www.jbc.org by guest, on January 11, 2010

'Lloyd,

K.O.

a n dK a b a t .E A . .v n p u b l l s k d

results.

Table 1. Chemical and PhysicalProperties of Reduced Oligosaccharfde Fractions Purified Preparative Paper Chromatography. Reduced Oligosaccharides Radioactivity Sample No. Yield cmposition gl cpm/ug Mol.
N

by BioGt1 P-2. Charcoal-celite Colmn and

Uolar ratio DGlCNAc OCal DGalNAc-ola, GlcWc, O b 1

Optical rotation
(o)& (a);&

Rf

ut.
1231 (1115) 1205 (1115) 1253 (1115) 1001 ( 953) 761 (750) a27 (750) 4.36 777 (750) 579 (588) 526
(588)

Analysis 8y HPLC

11.3 14.3

389
397.2

3.35

31.0

46.9

1:1.7(2):3.2(3) 1:1.9(2):3.5(3)

-31.1 -31.4

-108.7
-108.9

TWO peaks 1B and IC (Fig I S ) .

3.6151.3 33.7

Three major peaks ZA! 28. 6 2C (Fig

la).
TWO peaks 13A and

13 3

5.6
16.0

382.0 478

3.65 3.83

35.0 38.0

41.7 37.6

1:2.0(2):2.9(3) 1:1.8(2):2.1(2)

-20.0 -34.8

-80

13C (Fig. l a ) -107.2

withseveraltiny peaks. One major peak (48) and one small peak (4A). One peak. One major oeak (lac) one mino:' peak (Fig l b ) .

One major peak

32.0

629

3.51

25.0

54.9

1:0.95(1):2.5(2)

-49.4

-1556

5 16

13.9 5.5

578.9 616.2

3.47

24.0

47.2 33.9

1:0.91(1):2.2(2] 1:0.94(1):1.5(2)

-43.2 -42.7

-124.0 -146.0

26.0

dnd

6
7

19.3 43.2
9.9

827 909 894

4.1431.2 30.7 5.07

4.00

1:0.81(1):1.0(1) 33.0 31.8 1:0.85(1):0.97(1) 1:0.63(1):0.95(1)

-62.7

-169.5 -163.0 -154.5

One peak. One peak.

TWD peaks: one major 88, one minor 8A (Fig 1 ) .

35.5 25.9

-51.8 -57.9

ab

535.1

10.5

1088

440 (426) 3.83 377 (385)

6.19

40.7

2.1

1:0.88(1):0

-66.4

-171.7

One peak and

tiny

one

Peak.

10
29

16.6 6.1

1332 1198.5

45.3
50.9

1:0:0.93 1:0:1.2(1)

-53.0

-162.0
-162.0

One peak.
One peak.

f 385

399.5

3.19

0.41

-53.0

a Estimated by theextent
b unsaturation t e s t (+).

of labeling relative to that

of radioactive DGalNAc-ol ( 2 1 ) . values i n parenthesis

LR

theoretical.

7184

Blood Group Carbohydrate Structures

Downloaded from www.jbc.org by guest, on January 11, 2010

Fig. 2. Proposed structures and d i s t r i b u t i o n of oligosaccharidesisolated from U S 1 s t Smith degraded blood group A a c t i v e g l y c o p r o t e i n p u r i f i e d from human o v a r i a n c y s t f l u i d . Chemical and physicalpropertiesaregiven i n Table 11. Most of the Sampler p u r i f i e d by hplcare s t i l l mixtureswith several minor components detected by glc-ms. Hexasaccharides(6.1 mole X ) 2A ( R 1 ~ 51.17) 26 ( R 1 ~ 51.17) Trisaccharides (24.9 mole X ) GlcNAc minor 6 ( R fi 2.6, rs1.6 R L lO, . ~ \ ~ minor Gal61+3GalNAc-ol f r a c t i o n of sample 6 i l l u s t r a t e d by glc-m) G1 a (~1.4 GlcNAc (61.6 GalNAc--01 2C

Gal$1~3GlcNAc61JGal61+3GlcNAcn1+3Galn1+3GalNAc-ol

Galsl+3GlcNAcsl+3Galsl+3GalNAc-ol
ts1.6 GlcNAc t61.4 G1 a Gal rs1.4 GlcNAc (~1.6

Gal$1+3GlcNAc61+3Gal61+3GalNAc-ol
Pentasaccharides (4.2 mole X )

RIM^

1.17)

7 (RIM5 2.8); 6 major ( R 1 ~ 52.6 one o f two major fractionsseparated and i d e n t i f i e d by glc-ms) and 16C minor RIM^ 2.6, one of three minor fractions demonstratedbyglc-ms).
6 major RIM^ = 2.6 , one of tw major fractions separated and i d e n t i f i e d b y IC-ms) and 16C minor ?R 185 2.6. one o f three minor fractions resolved by glc-ms).

G1cNAcs1+3Galel~3Ga1NAc-ol
Gal (61.4 GlcNAc w1.6

GlcNAcslJGalsl~3GalNAc-ol
Tetrasaccharides (16.0 male X )

3C ( R 1 ~ 51.5)

GalslJGlcNAc6l~3Gal~l+3GalNAc-ol
Gal (61.4 GlcNAc 4B1.6 GaleldGalNAc-ol GlcNAc 4~1.3 Gal W1.4 GlcNAc W1.6 GaINAc-01

5 (RIM5 2.2R RL= 0.85) 48 (RIM5 1.73, 0.65). and 16C minor one o f three minor fractions separated and ident i f i e d b y glc-ms

G1 a (01.3 GlcNAc M1.6 GalNAc-ol G1 a (61.3 GlcNAc 461.6 (3-deoxy)GalNAc-ol

BE ( R 1 ~ 53.1)

E (RIM5 = 3.1) A

Disaccharides (15.4 male X ) GalslJGalNAc-ol GlcNAc ~1.6 GalNAc-ol 10 ( R L ~1.21. ~ RGal 1.0)
9B (Qat 1.1, R G 0.89) ~ ~

Previously isolated from 10

non Smith degraded blood group substances.

RG 0.92 (25). Horse RL 1.37,

Lewis RL 1.36 (7). JS RL 1.32 (7); Horse RG 0.87.

2X RL 1.64 (39):

RL 1.41 (25); T i j phenol insoluble R

1.68b,

T i j 2C%

Beach EP1 Ra Gl

0.87 (40); 06 R a Gl

0.87 (9).

6 minor: Lewis RL 0.86 (7); T i j 10%2X RL 0.62 (39). T i j phenol insoluble RL 0.85 (39).

48:

Lewis R,, 0.41 (19); Lewis RL 0.44 (7). JS RIM5 BP1 RL 0.42-0.46;

2.356 (7); T i j phenol insoluble RIM5 2.61 (39). T i j 1 %2X RIMS 2.76a. C 2.76b

(39); Beach

CG RL 0.44 (9).

96:

Beach P 1 R a Gl

0.88 (40).

Structures Group Carbohydrate Blood

7185

Hexasaccharide i s n w r s 2A. 13A 18, ZB

l C , 2C. 13C

(n.u.
43.4 44.9 43.7

1115)
1.0:2.2(2):3.3(3) 1.0:2.1(2):3.2(3) 1.0:1.8(2):2.8(3) -20.0 -27.0 -35.1

1073.2 3.31 (1115)

36.1 3.35 35.9

-83.3
-100.0
-119.0

1120.3 (1115)

1118.8 35.2 3.40 (1115) Pentasaccharide

R,&

3ca

1.5

949

3.83

38.0

37.6

1.0:1.9(2):2.1(2)

-34.8

-107.2

Tetrasaccharide
5

827 (750)

3.47

24.3

47.2

1 .0:0.82(1):1.9(2)

-43.2

-124.0

4.5

1.1

1.8

4Bb 16CC(ma~or) 763 25.4 (750) 4.2

48.3 32.5

0.23 ,0:1.0(1):2.4(2) .0:0.9(11:1.4

4.9 -39.0 -55.0

-150.0 -181.0

2.2

Trisaccharlde gd 7e 8Bf 8A' 4.14 579.0 (588) 30.7 5.07 35.5 31.2 1. 0 : 0 . 9 ( 1 ) : 1 . l ( 1 ) 0.21 1.0:0.83(1):1.0(11 1.0:0.52(1):0.95(1) 1.0:0.84(1):1.76(2) -62.7 4.4 -51.8 -37.0 -22.5 -169.5 -163.0 -191 .O -70.0 4.3 1.3
0

Downloaded from www.jbc.org by guest, on January 11, 2010

526.0 ( 588 )

33.0

2.1

572.2 24.43.9 (578)

35.8
39.7

368(?)*

3 . 3 ( ? ) 24.0

Disaccharide

9
10

454.3 (4261 397.4 (3851

6.2
3.83

40.7

2.1 45.3

1.0:0.88(1): 1.0:0:0.9(1)

-66.4

-171.7 -162.0

3.0
4.3

1.1 1.9

0.25
1.0

-53.0

+ Estimated by r a d i o a c t i v e DGalNAc-ol

(Zi), ' u n s a t u r a t e dt e s tp o s l t l v e
as i n F i g .
2.

a t o f i n d l c a t e Ho. o f s u b f r a c t i o n found byglc-ms a One main component and one m a l 1 peak. One main componentand One major and two minors.

e M l x t u r e o f one major and several minors A major component

++ HolarRatio
t

2 minor bigger

components.

three minors.
(Two majors and one m i n o r )

M i x t u r e so ft h r e et r i s a c c h a r i d ei s o m e r s

Structures Group Carbohydrate Blood

7186

Figure 8. derivative.

Mass ~ p e c t r mO f Sample 5 as the permethylated

and N-triflUOTOaCetylated

Figure 5. Mass spectrum of trisaccharide a l d i t o l found m Sample 6 as the pernethylated and N-tTiflUOTOdCetyldTed d e r i v a t i v e .

Sample 48 contained 1 branched tetralaccharide alditol Gal t81.4 GlCNAc +81.6 cIl11~3GalNAc4l

whichcould

be i d e n t i f l e d as:

Downloaded from www.jbc.org by guest, on January 11, 2010

The r e l l t l v e p s i r e i i d u e r mlt be l i n k e d to t h e 3- and 6 - p s i t i o n r O f t h e GdlWc-01. t i a n s o f s u b s t i t u t i o n Of the 61lNLc-01 bv these residues muld sot be d t t e r n l n e d f l o n t h e

1111.

Figure 6. Mass spectrum o f t r i s a c c h a r i d eI l d i t o l and h t r i f l u a r o a c e t y l a t e d d e r l r a t i v t .

found i nS a w l a

6 I S t h ep e r m t h y l a t e d

S a w l e 8A was f o r d as a result of Peeling by e l i m i n a t i o n o f t h e w b s t i t u t e n t i n t h e the GalNlc residue forming a 2.3 u n s a t u r a t e d e r i v a t i v eT h i e n l i n e . s I-porition f o d e r i v a t i v e *hen p a r t i a l l yr e a r r a n g e d t o an imine and subsequently reduced f m r 3-deoxyA t r i s a c c h a r i dled i t o l w l d l C be i d e n t i f i ebg l c 4w i t h dy S I probable GalNAc-3I. StVUCtUre Of:

6a181~361cW~ld(3-dcory)bal~4l
Periodate Oxidltion data on sample 6 which was il mixturethree of incmsirtent with this structure. i s m s were n o t

Stru~turalI n a l y s i s of T e t r a r a c c h a r l d e A l d i t o 1 r - W branched and two linear tetrasaccharldealditolscouldbeidentifiedby9lc-Mof fhe P e m t h y l a t e d and C t r i f l u o m a c c t y l ated derivatives. The mass Spectr~m Of S W l e I (Table 11) i s s h m i n Fig. 7. The Aand +series of ions nlr 314. m l z 518. nlz 817. mlz 330 md m/z 629 g i v e a sequence of hexNTFhex-hexNlFhexl(rF-31. m e hexNTF-htx l i n k a g ei sd e t e n i n e d t o be I J by the abundance Of I12 187 as diSCcylled for t h e t r i s a c c h a r i d e a l d i t o l s . The linkage bct~eHl h e hex t and the i n t e r n a l hexNTF C m m t be deteminined frm the mass s p c t r u : h a e v e r . the presence of a d-&rubrtituted 6 I c W in t h e l e t h y l a t i o n a n a l y s i s sh-d t h a t t h e hexdexNTFlinkage w s t be- 1 4 . me ions m/z 228 end nlz 272 shw. I S pIeviously discussed. that the hexNTF-1 i s6 4 s u b r t i t u t e d . Baled M t h e abDW data and O p t i c a ll W t a t i Mt h ef o l l w i n g i s the r t r u c t u r F f w smple 16C:

GI a

inl.4
GlCNAC

61cW1lr)6al~lr(61c~#ld6alW-31

W.6

G~lnlr3G1~NA~8lJGalsl~3GalNAc-ol

GalNk-01, GlCNAC, and Gal l n the ratlo 1:2:3. Sampler 18 and 28 a l s o contained d both m t h y l a t i o n analysisrevealed 3 . 6 - d i - ~ l ~ b l t i t ~ t eGal, non-reduclng t e r n i n d l Gal, and 4 - & w b s t i t u t e d GIcNAc and 3-&1ubstltuted GalNAC-01. Since 3-O-substituted only GalNAC-Ol was found. i t can be aiiumd that previously the described tefrdraccharlde alditol:

The 3-

tal8lt3Gl~NA~8lt3Gal1lr3GalNAc-ol

I
Gal .~1.4
GlCNAC

M*

+1,6

GdlnIr3G1cNA~~1~3G~lslr3GalNAC-01