Immunochemical studies on blood groups: Structures and immunochemical properties of nine oligosaccharides from B-active and non-B-active blood group substances of horse gastric mucosae

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 172. 535-550 (1976)

lmmunochemical Studies on Blood Groups

Structures and lmmunochemical Properties of Nine Oligosaccharides from B-active

and Non-B-active Blood Group Substances of Horse Gastric Mucosae.l* 2

WALTER NEWMAN AND ELVIN A. KABAT

Division of Chemical Biology and the Departments of Microbiology, Human Genetics and Development, and Neurology, College of Physicians and Surgeons, Columbia University, and the Neurological Institute,

Presbyterian Hospital, New York, New York 10032

Received July 3, 1975

Oligosaccharides from baseborohydride-treated B-active and non-B-active glycoproteins of horse stomach mucosae were purified chromatographieally on Bio-Gel P-2, charcoal-Celite, paper and high pressure liquid chromatography. From calorimetric and gas-liquid chromatographic analyses, methylation, quantitative periodate oxidation and Smith degradation, structures of nine oligosaccharides are proposed. Seven have not been previously described. The oligosaccharide isolated in largest amount in the B-active reduced tetrasaccharide analogous to an A-active reduced oligosaccharide from pig submaxillary mucin, and a reduced octasaccharide, the largest isolated, has two B determinants and may represent full expression of B-specific biosynthetic potential of the mucosal lining. Three B-active and one non-B-active oligosaccharide possessed the core structure previously identified in oligosaccharides from human blood group, B, HLeh, Lea and precursor I substances. Two non-B-active and one B-active compound inhibited the cross reaction of type XIV horse antipneumococcal sera with blood group substances. Terminal nonreducing a-linked DGIcNAc (n-2-acetamido-2-deoxyglucopyranose), previously found in oligosaccharides of hog blood group substances, was also present in a tetrassarcharide of the non-B-active material. Oligosaccharides released from blood group glycoproteins of horse stomach mucosae are smaller and hence less heterogeneous than those from human ovarian cyst and perhaps hog A + H and human gastric mucosae.

The existence of species differences in A- B- and H-active blood group substances

horn hog, horse, cow and humans has been well established by immunochemical analyses and chemical composition (l-4). Such differences can be an important guide to the elucidation of the carbohydrate structures responsible. For example, the detec- tion in hog mucin A and H substances of a

’ Supported by grants from the National Science Foundation, No. BMS-72-02119A02 and 32543 X-l, and a Program Project Grant from the National Institutes of Health, No. 5P0 GM 18153-05.

2 From Part III of a dissertation submitted by W. Newman in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Faculty of Pure Science, Columbia University, New York. This is article No. LX1 of a series; the previous article appeared in Arch. Biochem. Biophys. 172, 524-534.

new antigenic determinant reacting with Con A (5, 61, goat antisera to hog H gastric mucin (7) and human antisera to hog A gastric mucosa (8) led to the isolation of oligosaccharides possessing terminal nonreducing a-linked DG~cNA$ (5, 6).

3 Abbreviations used: N, nitrogen; GalNAc, 2- acetamido-2-deoxygalactopyranose; GlcNAc, 2-acetamido-2-deoxyglucopyranose; Fuc, 6-deoxygalactopyranose; Gal, galactopyranose; Glc, glucopyra- nose; glc, gas-liquid chromatography; hplc, high pressure liquid chromatography; R,;, R,, and R,,,, migration distances in paper chromatography relative to galactose, lactose and isomaltopentaose, respectively; R, a mixture of 3-hexenetetrols; AbN, antibody nitrogen, lo],,,,,, maximum ellipticity; AC, mono-O-acetyl; AC,, di-0-acetyl; Me, mono-O- methyl; Me,, di-O-methyl; etc.; CD, circular dichro- ism; ORD, optical rotatory dispersion, [ml, molecular rotation; Con A, concanavalin A.

535 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

536 NEWMAN AND KABAT

Hemagglutination inhibition and quantitative precipitin assays may not reveal all species specificities of blood group substances since the antibodies and lectins used generally require accessible determinants. The precise structural analysis of oligosaccharides released in undegraded form by base-borohydride treatment can establish species specificities or similari- ties directly. Because of the extensive heterogeneity and limited quantities of blood substances, only those oligosaccharide chains present in considerable amounts can be fully characterized. Evidence was presented in Ref. (9) that horse blood group glycoproteins contain shorter oligosaccharide chains than do those of human (10,111 or hog (121.

This investigation records the structural and immunochemical characterizations of nine reduced oligosaccharides released from horses B-active and non-B-active materials by NaOH-NaBH, under conditions which do not cause peeling from the reducing end. In addition to N-acetyl-ngalactos- aminitol, two of the oligosaccharides have been previously isolated from both human (11, 13) and hog (14, 46) blood group substances. From non-B-active horse mucosal glycoproteins three oligosaccharide structures are proposed, one a trisaccharide thus far unique to horse, the second a tetrasaccharide analogous to that proposed for the linkage region to the peptide backbone of hog mucosal glycoproteins (121, and a third containing a fucose substituted on a core tetrasaccharide structure attached to the peptide in human ovarian cyst blood group substances (10, 11, 13, 15). From the B-active sample four oligosaccharides that inhibit B anti-B precipitation have been isolated, including a tetrasaccharide in largest amount and an octasaccharide with two B determinants that more completely expresses the biosynthetic potential. Both compounds have recently been isolated in this laboratory from a human ovarian cyst with B and I activities (16). B- active compounds were not isolated from non-B material.

MATERIALS AND METHODS

Nitrogen, methylpentose tfucose), hexose (galactose), hexosamines, N-acetylhexosamines and galae-

tosamine were determined calorimetrically (17-191. In addition, galactose, N-acetylglucosamine and N- acetyl-n-galactosaminitol were quantitated after methanolysis and 0-aeetylation by glc as described in (9). Inositol was used as an internal standard.

Quantitative periodate oxidation was performed on 0.5 wmol of sample. Consumption of periodate was determined by an iodometric titration method (201, formic acid released, by addition of excess NaOH and back titration with HCl(211, and formaldehyde, with chromotropic acid (17).

Smith degradation (22) was carried out by using the periodate form of Rexyn 201 (231, 200-400 mesh (Fisher Scientific Co.) and analyzing for degradation products by glc (11).

Methylation of 1 mg of oligosaccharide was performed (7, 24) by using N,N-dimethylformamide and methyl iodide with BaO as catalyst. After methanolysis, the partially methylated methyl glycosides were analyzed by glc at 100°C (stainless-steel columns) or 120°C (glass columns) on ECNESS-M and 143°C (stainless-steel) on NPGS and as their acetate derivatives at 140°C (stainless-steel) or 160°C (glass) on ECNSS-M or at 193°C on NPGS. The low tem- peratures were used to identify neutral sugars by reference to methyl 2,3,4,6-Me,oGlc and high tem- peratures for identification of aminosugar derivatives by reference to methyl 3,4,6-Me,nGlcNAc. The standard methylated sugars used in identification have been described previously (25). Some variation from one run to another was noted in the relative retention time for methyl 4-Ac-3,6-Me,- DG~cNAc. Values obtained are indicated in parenthe- ses in Table IIb adjacent to peak values identifying this compound for each methylated oligosaccharide. R,,0.86 and RJ.36 (111 were used as standards for 3, 6-AC,-1, 4, 5-Me,-N-acetyl-o-galactosaminitol and 3- AC-~, 4, 5, 6-Me,-N-acetyl-o-galactosaminitol, respectively.

Tests for unsaturation were based on the ability of hexenetetrols to decolorize KMnO, and Brz (21).

Charcoal-Celite chromatography was performed on a 27 x 4.5 or 54 x 4.5cm column of Darco G-60 activated charcoal:Celite, 1:l (26, 27). Oligosaccha- rides were eluted by using a gradient of ethanol. Bio-Gel P-2 chromatography of oligosaccharides was performed as described (91. Analytical and preparative descending paper chromatography was done on S and S Green Ribbon in solvent l,l-butanol:pyri- dine:water, 35:39:26, or solvent 2,1-butanol:pyri- dine:water, 6:4:3. R values of oligosaccharides were measured relative to galactose (Ro) lactose CR,) or isomaltopentaose (R ,nS).

Reduced oligosaccharides were from the combined B-active or non-B-active pepsin- or Pronase- digested glycoproteins of horse gastric mucosae after treatment with 0.05 N NaOH in M NaBH, at 50°C for 16 h (9, 281. The first step in fractionation (9) was chromatography on Bio-Gel P-2. Further purifica-

STRUCTURES OF HORSE BLOOD GROUP OLIGOSACCHARIDES 537

tion, characterization and determination of the structures is the subject of the present report. In general, peaks eluted from different Bio-Gel P-2 columns were combined based upon position relative to the isomaltose oligosaccharides, optical rotation at 365 nm and presence or absence of methylpentose. Peaks eluted from charcoal-Celite columns of the same dimensions were pooled if they were indistin- guishable by paper chromatography, ethanol concentration required for elution and presence or absence of methylpentose. Rechromatography of materials from charcoal-Celite and paper on Bio-Gel P-2 was performed to remove inert materials.

High pressure liquid chromatography (hplc) was performed at room temperature on a Waters As- sociates Model ALCI401 liquid chromatography by using a series two 12 x l/4-in. columns of PBONDAPAKicarbohydrates. Depending on size or relative purity of the compound in question, flow rates offrom 0.5 to 2.0 ml/min were used, corresponding to pressures of from 750 to 2300 psi. The mobile phase was acetonitrile:water, 3:l. Peaks were pooled based on continuous monitoring of the ef- fluent by refractive index and absorption at 254 nm; hplc was used at the end of the fractionation proce- dure to resolve mixtures of oligosaccharides unresolved by the other methods and as an additional criterion of purity.

ORD and CD analyses were performed on a JASCO Model J-20 automatic recording spectropo- larimeter; 0.1 mgiml solutions of oligosaccharides were measured in a l-cm cell at 27°C.

/3-N-acetylhexosaminidase of Turbo cornutus was obtained from Seikagaku Fine Biochemicals (Tokyo, Japan). It was used as described (30) on 0.9 wmol of RL0.88 incubated for 5 days in citrate/phosphate buffer, pH 4.1. Aliquots were taken at intervals and analyzed for free N-acetylhexosamine. As a control, p-nitrophenyl-P-N-acetylglucosaminide was run si- multaneously.

Type XIV horse anti-pneumococcal serum was a 1939 bleeding of horse 635 (31, 32). Human anti-B serum 310, (4) was used as described (9, 28). Struc- tures of the standards used for inhibition of precipitation are shown in Fig. 3. Beach R,0.44, Beach R1.0.26 (15) and Lewis N-l RL0.44 (11) have been described. Quantitative inhibition of precipitation was carried out on a microscale (17). OG 20% from 2nd 10% (13) was used as antigen in the type XIV assay and all operations were carried out at 4°C (17). For B anti-B inhibition, horse 4, 25% (2) was used.

Infrared analysis of RL0.88 was kindly performed by Dr. Lajos Bandy on a Perkin-Elmer Model 521 grating infrared spectrophotometer.

RESULTS

After NaOH-NaBH,-catalyzed release of the carbohydrate chains from the pep-

tide backbone, neutralization, dialysis, desalting, elution from ion-exchange resins and a second desalting with Retardion, 11 fractions were obtained (9, Table II): A-F from B-active and a-e from non-B-active. Nine oligosaccharides plus N-acetyl-n-galactosaminitol were isolated and characterized. Their chemical properites are given in Table I along with results from periodate oxidation, and their structures are presented in Fig. 1. They are numbered, I, II, III-X in Fig. 1 and in the following description. When more than a single sample of the same oligosaccharide was isolated from either B-active or non-B-active materials, its entire structure was established but results are mentioned only in the footnotes to Tables I and II.

Oligosaccharides from B-Active Material

Compound Z (N-Acetyl-r+galactosamini- toll. Fractions A, B, C and D (9, Table II) each contained a small amount of material without methylpentose eluting from Bio- Gel P-2 between IM2 and IM4. These were combined (7 mg) and applied to charcoal- Celite. A major peak eluted at 2.5% ethanol which chromatographed as a single peak on Bio-Gel P-2 (1.8 mg) and was identified by paper chromatography as RJ.38. It gave a single peak on hplc.

Compound ZZ. Fractions A, D and E each showed a peak containing no methylpentose on Bio-Gel P-2 in the IM4-IM6 region. These were combined for charcoal- Celite chromatography and a major peak eluted at 6% ethanol giving one spot, RJ.41 (2.1 mg) on paper in solvent 2, and a single peak on Bio-Gel P-2 and hplc. Bio- Gel P-2 chromatography of fractions C, D and E gave material eluting close to RJ.41 which eluted from charcoal-Celite at 6.5% ethanol (6.9 mg). Repassage through Bio-Gel P-2 gave 3.8 mg of a compound of RJ.37 in solvent 2 which eluted on hplc as a single peak identical to RJ.41.

Compound IV. Fractions C, D and E showed a broad methylpentose-containing peak eluted from Bio-Gel P-2 in the IM4- IM6 region not completely resolved from adjacent material. Three fractions, a major central peak and two areas on either

cn

w 02

TABL

E I

YIEL

DS,

ANAL

YSES

, M

OLE

RATI

OS,

SP

ECIF

IC

OFTI

CAL

ROTA

TIO

NS

AND

RESU

LTS

OF

QUA

NTIT

ATIV

E PE

RIOD

ATE

OXID

ATIO

NS

OF

ISOL

ATED

OLIG

OSAC

CHAR

IDES

Oligo

- Nu

mbe

r sa

ccha

- rid

e

R,1.

37'

Calcd

R,

,0.6

7' Ca

lcd

R,1.3

8’ Ca

lcd

R,0.

65'

Calcd

R,

l.04 C&

d R,

O.&%

Ca

lcd

R,0.

69

Calcd

R,

,,1.1

5 Ca

lcd

Rw.J.

18

Celcd

R,

,,0.65

Ca

lcd

II 5.9

II 17

.1

III

17.6

IV

30.4

V 2.3

VI

6.4

VII

1.9

VIII

1.7

IX 2.1

X 4.1

Yiel

d (m

g)

Perc

ent

com

posit

ion

Mole

ra

tios”

Pe

rioda

te

oxida

tion”

O

ptica

l ro

tatio

n (d

egre

es)

N Fu

c Ga

l Gl

cNAc

N-

acety

l. ga

lactm

- am

initol

3.5

3.6

3.1

3.6

3.3

2.6

1.9

2.0

4.2

4.8

5.6

5.3

3.1

3.1

3.0

2.6

3.2

2.6

2.5

2.0 -

0.9

44.1

0.0

46.8

0.7

44.5

0.0

46.8

33.8

36.2

30.9

33.0

23.2

56.2

23.7

51.9

1.9

27.6

0.0

30.6

1.5

25.1

0.0

22.8

15.5

30.6

18.3

40.2

14.6

46.9

15.5

51.0

16.1

56.0

15.5

51.0

21.2

51.0

24.0

52.7

0.0

51.6

1.1

0.0

1.1

0.0

57.9

1.0

0.0

1.0

1.6

56.5

1.0

0.2

1.0

0.0

57.9

1.0

0.0

1.0

0.9

40.8

1.3

1.1

1.2

0.0

42.0

1.0

1.0

1.0

0.0

32.2

0.9

1.0

2.2

0.0

32.2

1.0

1.0

2.0

34.6

39.0

2.0

0.1

0.9

37.6

37.9

2.0

0.0

1.0

40.3

31.5

2.8

0.1

1.0

55.9

28.2

3.0

0.0

1.0

20.6

n.d.

” 2.4

1.0

1.8

24

.7 24

.9 2.0

1.0

2.0

15

.8 n.d

. 2.5

1.0

3.0

20

.9 21

.1 2.0

1.0

3.0

18

.7 m

d.

2.3

1.0

3.1

20.9

21.1

2.0

1.0

3.0

15.6

14.5

2.1

2.0

4.1

16.2

16.3

2.0

2.0

4.0

N Fu

c Ga

l Gl

cNAc

N-

acet

yl-

Perio

date

Fo

rmal-

Fo

rmic

gala

&s-

cons

umed

de

hyde

ac

id am

inito

l re

leas

ed

relea

sed

0.0

1.0

3.9

0.9

2.2

0.0

1.0

4.0

1.0

2.0

0.0

1.0

4.1

1.0

1.6

0.0

1.0

4.0

1.0

2.0

0.0

1.0

4.8

1.0

2.3

0.0

1.0

5.0

1.0

2.0

0.0

1.0

5.4

1.0

2.6

0.0

1.0

6.0

1.0

3.0

1.0

1.0

6.3

1.3

n.d.”

1.0

1.0

4.0

1.0

2.0

1.3

1.0

4.1

1.0

1.6

2.0

1.0

4.0

1.0

1.0

1.0

n.d.

n.d.

n.d.

n.d

. 1.0

1.0

6.0

0.

0 2.0

0.8

n.d

. n.d

. n.

d.

n.d.

1.0

1.0

7.0

0.0

3.0

0.8

n.d.

n.

d.

n.d.

n.d

. 1.0

1.

0 7.0

0.

0 3.0

1.2

1.0

5.9

0.4

3.7

1.0

1.0

9.0

0.0

4.0

-41

-133

-48

-161

l%

4

-139

-420

E -14

-49

-57

- 15

3 5

+22

+52

‘J:

is

-67

-216

z

-19

-66

-29

-100

-7 -37

(I M

oles

per

mol

e of

N-

acet

yl-D-

gala

ctos

amin

itol,

exce

pt

for

VII,

VIII,

an

d IX

, ex

pres

sed

as

mol

es

per

mol

e of

LF

UC.

Ir M

oles

per

mol

e of

ol

igos

acch

arid

e.

c R,

1.

41

and

R,;0

.92

were

sh

own

to

be

iden

tical

in

al

l re

spec

ts

to

R,

1.37

an

d R,

,0.8

7;

R,,0

.85

wasid

entic

al

to

R,,l.3

8,

and

RIO.

66

and

RIO.

68

were

id

entic

al

to

R,0.

65.

Yiel

ds

given

ar

e th

e to

tals

of

all

fract

ions

; R,

0.68

an

d R,

,0.8

5 bo

th

cont

ain

inert

weigh

t. ”

Not

done

.


;LI”C3 I pctuc

Pl 1 2 l x D~~111-35)GB151-3N-ace~/L-D-galscrosammlro,

%2 65

FIG. 1. Proposed structures of oligosaccharides isolated from B-active and non-B-active materials.

side were chromatographed separately on charcoal-Celite. In each, a peak with methylpentose eluted in the 13-15% ethanol region. Paper chromatography in solvent 2 showed them to be nearly identical: R,0.65, R,,0.66 and RL0.68. They were not combined but were eventually established as identical; total yield, 30.4 mg. Each gave a single peak when chromatographed on Bio-Gel P-2; hplc of R,0.65 yielded a single peak. RL0.65 is derived from peak 4 -in Fig. 2 of Ref. (9).

Compounds VIZ, IX and X. The compounds of highest molecular weight from fractions C, D and E eluted as a broad peak in the IM6-IM8 region and larger; partial separation of components was achieved by two fractionations on charcoal-Celite. R &. 18 (IX) eluted from charcoal-Celite at 17% ethanol while R,,,1.15 (VIII) and R,,,0.65 (X1 eluted together in the 20-22% ethanol region. Preparative paper chromatography in solvent 1 for 11 h yielded R,MJ.~S and RIMsO. and for 16 h


gave R&.18. Each was rechromato- graphed on Bio-Gel P-2, giving single symmetrical peaks; yield, 1.7 mg of R&.15, 2.1 mg of RIMr,l.18 and 4.1 mg of RIM;,0.65. Hplc of RIM31. 15 and R&.18 gave single peaks.

Oligosaccharides from Non-B-Active Mate- rial

Compounds I (N-Acetyl-Bgalactosamin- itoll and II. Fractions a, c and d gave peaks from Bio-Gel P-2 which eluted in the IM2-IM4 region. When combined and chromatographed on charcoal-Celite, they emerged as two peaks, one at 4% ethanol and the second at 7% ethanol. Paper chromatography of each in solvent 2 gave single spots, RJ.26 (I) and RG0.87 (II), respectively. Bio-Gel P-2 chromatography gave a single peak for Ro1.26 (2.3 mg) and RG0.87 (13.0 mg); hplc also showed single peaks. Ro1.26 is identical to RJ.38 isolated from the B-active material. Both are N-acetyl- D-galactosaminitol (I) and were purified from peak 1 as shown in Fig. 2 of Ref. (9).

Compounds II and 111. Fraction b gave a single symmetrical peak in the IM4 region of Bio-Gel P-2. It eluted from charcoal- Celite as two peaks, one at 6% ethanol without methylpentose and the other with methylpentose at 12.5% ethanol. Paper chromatography in solvent 2 showed the first peak to be Rc0.92 (II), the second RG0.85 (III). RG0.92 gave a single peak on Bio-Gel P-2 and hplc. It is identical to RJ.37 (II) and RJ.41 (II) isolated from the B-active and RG0.87 (II) from the non-B- active material. All were derived from peak 2 in Fig. 2 of Ref. (9) or the analogous peak from a different Bio-Gel P-2 column. RG0.85 eluted as a single peak both on Bio- Gel P-2 (2.6 mg) and on hplc. This compound was isolated from the non-B-active peak 3 in Fig. 2 of Ref. (9).

Compound III. Fractions d and e gave methylpentose-containing peaks eluted from Bio-Gel P-2 in the IM4 region. On charcoal-Celite each eluted at 20% ethanol and were combined and rechromat- ographed on Bio-Gel P-2 giving a single peak. Analytical paper chromatography in solvent 2 showed a single spot, RJ.38 (15.0 mg) identical with RG0.85 (III) (above).

Compound V. Material eluting in the IM6 region of Bio-Gel P-2 of fractions d and e were combined for charcoal-Celit,e chromatography. A major peak eluted at 6% ethanol which gave a single peak (2.3 mg) on Bio-Gel P-2. This compound in solvent 2 had RJ.04 and showed one peak by hplc.

Compound VI. Fractions a and c gave material from Bio-Gel P-2 eluting just after IM8. Each was applied to charcoal- Celite separately and eluted as a broad unresolved band between 13 and 15% ethanol. Preparative paper chromatography in solvent 1 gave a major component, R,0.88. Although it eluted as a single symmetrical peak from Bio-Gel P-2, it was resolved by hplc into one major (6.4 mg) and two minor peaks.

Compound VII. On Bio-Gel P-2, fractions d and e each gave material in the TM8 region eluting on charcoal-Celite at 23-25% ethanol. Analytical paper chromatography of each showed a single spot, R,0.69. These were combined and gave a single peak on Bio-Gel P-2 (1.9 mg).

Determin,ation of Structures

Analytical data, specific optical rotation of 589 and 365 nm and results from quantitative periodate oxidation are shown in Table I. All oligosaccharides were tested for unsaturation and found negative. Glc identifications of the methylated, methanolyzed and 0-acetylated oligosaccharides are shown in Tables IIa and IIb. Results of quantitative inhibition of B anti-B and type XIV precipitation are given in Fig. 2. Figure 3 shows the structures of the standards used in the inhibition assays.

Compound I. RJ.26 from the non-B and RJ.38 from the B material were isolated in small amounts and identified as N-acetyl-D-galactosaminitol after a final purification by hplc. Methanolysis and O-acetylation followed by glc revealed only N- acetyl-D-galactosaminitol. Quantitation by glc allowed for a correction for inert weight in RJ.38 [aID values for RJ.26 (-46”) and Ro1.38 (-35”) were close to the standard N-acetyl-D-galactosaminitol (-40.2”) (13).

Compound II. Rc0.87 and RG0.92 (combined yield 17.1 mg) from the non-B-active and RJ.37 and RJ.41 from the B-active

TABL

E IIa

GAS

CHRO

MAT

OGRA

PHIC

ID

ENTI

FICA

TIO

N OF

TH

E M

ETHY

L ET

HERS

OF

HE

XOSE

S FR

OM

PERM

ETHY

LATE

D OL

IGOS

ACCH

ARID

ES

: 2 Sa

mpl

e Nu

mbe

r Ti

me

of elu

tion

relati

ve

to m

ethyl

2,3,4,

6-tet

ra-0

-meth

yl-an

gluco

pyra

nosid

e Ide

ntity

of m

ethyl

glyco

sides

E

ECNS

S-M

” NP

GS”

% RJ

.37’

II 1.3

0 1.2

6 R&

87'

2,3,4.

6-M

e,-ffia

l

II 1.

31

1.22

8

R,1.

3Bc

2.3,4.

6-M

e,-r&

al

III

0.44

0.

47

2.84

R,

,O.6

5' 4.

53

2,3,4,

-Me,-

~Fuc

; 3,

4,6-

M.+

,-oGa

l IV

1.3

0 1.3

1 2,

3,4,

6-M

e,-~

Gal

8 RJ

.04

V 1.3

8 1.2

4 R,

.O.8

8 VI

2,3

,4,6-

Me,-

nGa1

2.8

1 3.

90

4.27

2.1

5 2.

75

3.05

R,,0

.69

VII

2,3,6-

Me,-

nGa1

m

0.47

1.

35

3.90

0.

48

1.26

2.90

4.

60

2,3,4-

Me,-

r.Fuc

; 2,3

,4,6-

Mea

-&al;

5;

R,,,1

.15

VIII

3,4,6-

Me,-

nGal

1.30

0.

47

1.24

8

R,~J

.lB

IX

2,3,4-

Me,-

LFuc

; 2,3

,4,6-

Me,-

&al

1.23

0.

59

1.21

R,M

,‘J.‘~

~ X

0.46

2,3

,4-M

e,,-r.

Fuc;

2,3,4,

6-M

e,-&a

l 1.3

7 0.

44

1.23

8

Refer

ence

co

mpo

unds

2,3

,4-M

e,,-I&

C;

2,3,4,

6-M

e,-nG

al

Meth

yl 2,3

,4,6-

Me,-

tile

1.0

1.0

s

Meth

yl 2,3

,4,6-

Me,-

nGa1

1.3

2 1.

24

Meth

yl 3,4

,&M

e+Ga

l 3.

78

2.91

4.52

?

Meth

yl 2,3

,6-M

e,-nG

a1

2.86

3.

92

4.31

2.13

2.74

3.

03

3 M

ethyl

2,3,4-

Me,-

r.Fuc

d 0.

48

0.49

0 $

” A

copo

lymer

of

ethyle

ne

glyco

l su

ccina

te po

lyeste

r an

d a

nitrile

sili

cone

po

lymer

(3%

) on

Ga

s Ch

rom

Q

(100

-120

mes

h).

b Neo

penty

l gly

col

succ

inate

(2%)

on

acid-

wash

ed

and

silaniz

ed

Chrom

asorb

W

(80

-100

mes

h).

z

c RL

1.41

and

R,j0.

92we

resho

wntob

eiden

tica1

ina11

respe

ctsto

R,.1.

37an

d R,

,0.8

7;

R,0.8

5was

identi

calto

R,

1.38;a

nd

R,0.6

6and

R,

0,68w

ereide

ntica

lto

R,1)

.65.

d

This

stand

ard

was

not

avail

able,

va

lues

are

taken

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m

a pre

vious

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. z E w W

I


TABLE

GAS CHROMATOGRAPHIC IDENTIFICATION OF METHYL ETHERS OF DGal,

OLICOSAC

Sample Number Times of elution relative to methyl-2-acetamido-2-deoxy-3,4,6-t& O-methyl-

ECNSS-M

RJ.37 Ra0.67 R ,.I.38 R,,0.65

RJ.04

R,.0.86

R,,0.69

R,MJ1.15

R,J.lB

R1~8.65

Reference compounds Methyla-3,4,6-Me,-

nClcNAc Methyl 4-Ac-3,6,-Mez.

DGIcNAc Methyl 4-Ac-3,6-Me,-

N-Me-DGlcNAc Methyl &~-AC,-

4,6-Me,-&al N-I R,.1.36 (11) N-l R,O& (11)

II 1.06

Ii: 1.05 1.05 IV 0.35 0.50 1.06 0.40

V 1.04 1.65 2.21 3.05 (2.98)

VI 1.00 1.06 1.65 2.16 2.84 (2.84)

VII 0.81 1.12 1.64 2.19 2.90 (2.84)

VIII 0.29 0.43 0.77 1.63 2.16 3.08 (2.98) 0.38

IX 0.33 0.46 0.81 1.10 1.54 2.08 2.50 (2.52) 0.35

X 0.28 0.43 0.79 1.06 1.57 2.12 2.80 (2.79) 0.44

1.00

+ (above)

1.10 1.58 2.08

0.35 0.46 0.39

1.02 0.86

(combined yield 5.9 mg) materials were all Compound III. R,,0.85 and RJ.38 (com- identified as DGalpl += 3N-acetyl-D-galac- bined yield 17.6 mg) isolated from the non- tosaminitol. Each contains equimolar B-active material were each shown to be amounts of galactose and N-acetyl-D-galac- LFuccYl+BDGalpl+=3N-acetyl-D-galactosa- tosaminitol. On periodate oxidation each minitol. The corresponding fraction from produced close to the theoretical number of the B-active sample was lost. They con- moles of formaldehyde and formic acid per tain equimolar amounts of fucose, galac- mole of compound and consumed the ex- tose and N-acetyl-D-galactosaminitol. pected 4 mol of periodate per mol. Smith Each consumed the theoretical amount of degradation of RG0.87 yielded glycerol and periodate and released the expected N-acetylthreosaminitol, the latter confirm- amount of formaldehyde and formic acid atory for a 1+3 linkage to N-acetyl-D- per mole of compound. Methylation anal- galactosaminitol. Methylation analysis of ysis showed methyl 2, 3, I,-Me,LFuc, each revealed methyl 2, 3, 4, 6-Me,DGal methyl 3, 4, 6-Me,DGal and 3-AC-~, 4, 5, 6- and 3-AC-~, 4, 5, 5-MeJV-acetyl-Dgalactos- Me&-acetyl-D-galactosaminitol. R ,0.85 aminitol. The specific optical rotatio.rs has about 23% inert weight. RJ.38 was were close to the value of N- 1 R J .36 ( - 53” obtained in crystalline form, mp, 249- at 589 nm and -174” at 365 nm) (11) and 251°C. The specific optical rotation of the the value of -48” at 589 nm reported ear- crystalline compound (Table 11 was lier (461. slightly more positive than values re-

STRUCTURES OF HORSE BLOOD GROUP OLIGOSACCHARIDES

IIb

DG~cNAc AND N-ACETYL-D-GALACTOSAMINITOL FROM PERMETHYLATED CHARIDES

543

aDghxopyranoside

NPGS

0.97 1.00 0.98

0.53 1.02

0.99 2.45 (2.44)

0.96 1.06 1.17 1.51 1.88 2.23 (2.21)

0.78 1.15 1.57 1.89 2.40 (2.39)

0.53 0.84 2.08 (2.03)

0.46 0.85 1.09 1.42 2.07 2.40 (2.39)

0.55 a.81 1.17 1.42 1.88 2.04 (2.03)

1.00

1.23 1.50 1.87

0.51

0.97 0.86

Identity (as methyl glycosides or as derivatives of N-acetyl-o-galactosaminitol)

3-Ac-1,4,5,6-Me,-N-acetyl-r+galactosaminitol 3-Ac-1,4,5,6-Me,-N-acetyl-o-galactosaminitol 3-AC-1,4,5,6-Me,-N-acetyl-D-galactosaminitol 2,3-AC,-4,6-Me,-oGa1; 3-Ac-1,4,5,6-Me,-

N-acetyl-o-galactosaminitol 3-Ac-1,4,5,6-Me,-N-acetyl-o-galactosaminitol;

4-Ac-3,6-Me,-N-Me-oGlcNAc; 4-Ac-3,6-Me,- oGlcNAc

3,4,6-Me,-DGlcNAc; 3-Ac-1,4,5,6-Me,-N-acetyl- n-galactosaminitol; 4-Ac-3,6,-Me,-N-Me- DGIcNAc; 4-Ac-3,6-Me,-DGlcNAc

3,6-AC,-1,4,5-Me,-N-acetyl-o-galactosaminitol; 4-Ac-3,6-Me,-N-Me-DGlcNAc; 4-AC-~,& MeroGlcNAc

2,3-AC,-4,6-Me,oGal; 3,6-AC,-1,4,5-Me,- N-acetyl-o-galactosaminitol; 4-Ac-3,6-Met oGlcNAc

2,3-AC,-4,6-Me,-oGa1; 3,6-AC,-1,4,5-Me,- N-acetyl-u-galactosaminitol; 4-Ac-3,6-Me%- N-Me-oGlcNAc; 4-AC-3,6-Me,-oGlcNAc

2,3-AC,-4,6-Me,nGal, 3,6-AC,-1,4,5-Me,- N-acetyl-wgalactosaminitol; 4-Ac-3,6-Me,- N-Me-oGlcNAc, 4-Ac-3,6-MeroGlcNAc

ported previously for this compound (lll:[a], = -124” and l&S = -382”, and from Ref. (46): [al,, = -114.7”, as would be expected.

Compound IV. R,,O.65, REP.66 and R,,O.68 are identical and have the structure shown in Fig. 1. Each analyzed for Fuc, Gal and N-acetyl-n-galactesaminitol in a ratio of 1:2:1. Periodate oxidation results were as expected except for the somewhat low value for periodate consumed for R ,.0.65 and R ,.O.SS. Methylation analysis of each revealed methyl 2,3,4-Me,~Fuc (except for R,*0.65 for which this volatile compound had probably evaporated before analysis), methyl 2,3,4,6-Me,uGal, methyl 2,3-AC,-4, 6-Me,nGal and ~-AC- 1,4,5,6 - Mea - acetyl -D - galactosaminitol. From these data the LFUC could be in either the C-2 or C-3 position. Quantita-

tive inhibition of precipitation (Fig. 21 showed all to be inhibitors of B precipitation and equally good on a molar basis. Hence the LFUC is assigned in o-linkage to C-2 and the nGa1 is assigned in a-linkage to C-3. Glc analysis of the Smith degraded products of RL0.65, 0-acetylated only, showed glycerol; the methanolyzed and O- acetylated products revealed galactose and N-acetylthreosaminitol as well, supporting the structure shown in Fig. 1.

Compound V. R J.04, isolated only from the non-B-active substance, was shown to be nGalpl~4nGlcNAcpl~3N-acetyl-n- galactosaminitol by the following criteria. It contained Gal, GlcNAc and N-acetyl-n- galactosaminitol in equimolar amounts. Methylation analysis gave methyl 2,3,4, 6-Me,uGal, methyl 4-Ac-3,6-Me,nGlcNAc, methyl 4-Ac-3,6-Me,-N-MenGlcNAc and

0 Me,nGlcNAc, methyl 4-AC-~, 6-Me,n- GlcNAc, methyl 4-AC-~, 6-Me,-N-Mea GlcNAc, 4-Ac-1,4,5,6-Me,-N-acetyl-n- galactosaminitol and methyl 2,3,6-Me,n-

FIG. 2. Inhibition by oligosaccharides or precipi- Gal. Smith degradation on a portion tation of (A) human antiserum to horse blood group subjected to 0-acetylation showed free B substance (310,) and horse 4,25% and (B) type XIV glycerol and N-acetylthreosaminitol. After antipneumococcal horse serum H635’38 and OG 20% methanolysis and 0-acetylation, DG~cNAc from 2nd 10%. was also found. The free N-acetylthreosa-

pco I minitol could only result if the sugar linked to the N-acetyl-D-galactosaminitol was destroyed by periodate. This had to be nGa1, since the 4-substituted DG~cNAc would be resistant to periodate and would remain glycosidically linked to N-acetylthreosaminitol. The presence of two kinds of DG~cNAc from methylation analysis, in roughly equal proportions, supports a tetrasaccharide structure (Fig. 1) despite the finding of only 1.3 mol of DG~cNAc per mol of N-acetyl-n-galactosaminitol. The tetra-

FIG. 3. Structures of standards used in inhibition saccharide structure is also consistent assays of type XIV and B anti-B precipitation. with the analytical data showing 21.6%

~-AC- 1,4,5,6,-Me,N-acetyl-n-galactosa- gala&se (theory, 22.8%), 31.5% N-acetyl-

minitol. Smith degradation (Rexyn) de- n-galactosaminitol (theory, 28.2%) and

stroyed the terminal galactose and the N- 5.6% N (theory, 5.3%) The low value for

acetyl-n-galactosaminitol but left the DG~cNAc of 40.3% (theory, 55.9%) is a con-

DG~cNAc intact. Products were N-acetyl- sequence of a ~GlcNAccul+4~GlcNAc

threosaminitol and glycerol, the former in- structure at the nonreducing end (33-36).

dicative of a 1+3 linkage to the N-acetyl- With methyl (Y- and ~DG~cNAc as model

n-galactosaminitol, the latter from the non- compounds (34) is has been shown that the

reducing gala&se. R J.04 was equal to rate of acid hydrolysis for the p compound is some 16 times faster than the rate for

the standard N-l R,0.44 on a molar basis in inhibition of type XIV precipitation. Mi-

the cy compound. Quantitative periodate

croscale periodate oxidation, omitting the oxidation gave 1.0 mol of formaldehyde (theory, 1.01, 1.6 mol of formic acid (theory,

analysis for formic acid due to insufficient 1.0) and consumed 4.1 mol of periodate material showed 6.3 mol of periodate con- (theory, 4.0) per mol of compound, the high sumed (theory, 4.0) and 1.3 mol of formal- formic acid perhaps being due to an inter- ___ _ _ dehyde released (theory, 1.0). The high nal Cannizzaro reaction (37, 38) by action

NEWMAN AND KABAT

value for periodate consumed is probably due to an imprecision in concentration; after hplc only 1.0 ml of an analytical solu- tion of 500 H/ml was available for periodate analysis. Data from calorimetric, gas-liquid chromatographic (Table I) and Smith degradation analyses support the structure in Fig. 1.

Compound VI. R,0.88, isolated only from the non-B-active materials, analyzes for Gal, GlcNAc and N-acetyl-n-galactosaminitol in a ratio of 1.0:1.3:1.0. Methylation analysis gave methyl 3,4,6-


of the NaOH on the periodate-oxidized oligosaccharide. This tetrasaccharide does not inhibit type XIV precipitation (Fig. 2); 500 nmol gave about 80-90% inhibition of the precipitation of 8.1 pg of Con A N by 28.5 pg of mucosa 7, 10% 2x fraction (cf. 61, approximately equal on a molar basis to methyl a-DG~cNAc. The a-linkage of the terminal DG~cNAc is assigned based upon its positive optical rotation, [(Y],) = +22”, its resistance to acid hydrolysis, inhibition of precipitation by Con A and data from ORD and CD analyses (Fig. 4, Table III). In addition it was not split by P-N -acetylhexosaminidase (a-N-acetylhexosaminidase was not. available). Infrared analysis

Frc. 4. ORD and CD spectra of three oligosaccharides from non-B-active horse blood group substance. Structures are given in Fig. 1.

showed no band at 1240 cm-‘, characteristic of sulfate.

Compound VII. R,,O.69, isolated from the non-B-active, analyzes for Fuc, Gal and GlcNAc in a ratio of 1.0:1.8:1.0. Meth- ylation analysis gave peaks for methyl 2,3,4 - Me+Fuc, methyl 3,4,6 - Me,nGal, methyl 4 - AC - 3,6 - Me,N - MenGlcNAc, methyl 4-AC-3,6-Me,nGlcNAc and 3,6- AC,-1,4,5-Me&-acetyl-n-galactosaminitol. This compound inhibited type XIV precipitation, but was slightly less active than the Lewis standard N-l R ,,0.44. Since this compound was unable to inhibit precipitation of the Lotus lectin (39), the LFUC is assigned in a-linkage to the DGal which is linked /31+3 toN-acetyl-D-galactosaminitol. Had the Fuc been on the other Gal there would have been no inhibition of type XIV precipitation and it would have been a good inhibitor of the Lotus lectin. The proposed structure is shown in Fig. 1. Because insufficient material was available for glc quantitation of N-acetyl-D-galactosaminitol and for quantitative periodate oxidation, the structure proposed must remain tentative.

Compounds VZZZ and IX. R&.15 (VIII) and R ,&. 18 (IX) were isolated only from B-active material. Per mol of LFUC, each

TABLE III

ORD AND CD PARAMETERS OF OLIGOSACCHARIDES ISOLATED AND OF REFERENCE COMPOUNDS

Compound

R,;0.87 (II) DGalPl -+ 3N-acetyl-D-galactosaminitol

RJ.04 (V) DGalPl + 4~GlcNAcfll+ 3N-acetyl-wgalac-

tosaminitol R,P.88 (VI)

~GlcNAcal -+ 4~GlcNAcpl + 4DGalpl -+ 3N-acetyl-wgalactosaminitol

Methyl cx~GlcNAc (40)

Methyl ~DG~cNAc (40)

DGalBl 4 4~GlcNAc (41)

DGalpl + 3~GalNAc (6) N-acetyl-D-galactosaminitol (16)

-6,397 None

- 16,235 220

-5,896

-3,100

-2,690

-2,860

-1,740 -4,496

223 +409

220

220

218

None None

-1,125

-1,976

+1,170

-552

+80

+500 -620

-5,272

- 14,259

-5.577

+ 1,930

-2,140

-2,940

-2,240 -3,876

-7266 (200)

-23,294 (208)

-23,048 (210)

-4200 (210)

-6300 (215)

-5000 (218)

-4424 (208)


analyzes for 3.0 or 3.1 mol of DGal (theory, 3.0),2.5or2.3molofN(theory,2.0)and0.8 mol of DG~cNA~. Insufficient material was available for quantitation by glc of N-acetyl-n-galactosaminitol or for quantitative periodate oxidation. Methylation of each gave peaks corresponding to methyl 2,3,4,- 6-Me,nGal, methyl 4-Ac-3,6-Me,nGlcNAc, methyl 4 -AC - 3,6 - Me, - N - MenGlcNAc, methyl 2,3-AC,-4, 6-Me,nGal and 3,6-AC,- 1,4,5 - Mea - acetyl - D - galactosaminitol. RIM51. 15 gave a peak corresponding to methyl 2,3,4-Me+Fuc, while R&.18 did not presumably because this volatile compound had evaporated before analysis. RIM51.15 had eluted from charcoal-Celite at 20-22% ethanol, and R&.18 eluted at 15-17%. They differed as well in their specific optical rotations (Table I) and in inhib- itory activity with anti-B and anti-type XIV. R&.15 is a good inhibitor in the type XIV system while RIM31.18 is rela- tively inactive. R&.18 is a strong inhibitor of B precipitation while RIM31.15 is weak and equal, over the limited range studied, to R,,0.65. The structures proposed are tentative in view of the limited characterization.

Compound X. R,,,0.65 was isolated only from the B-active material and analyzes for Fuc, Gal, GlcNAc and N-acetyl-n-galactosaminitol in a ratio of 2.0:4.1:1.2:1.0. Methylation gave methyl 2,3,4-Me,LFuc, methyl 2,3,4,6-Me,nGal, methyl 2,3-AC,- 4,6-Me+Gal, methyl 4-Ac-3,6-Met-N-Me- DG~cNAc, methyl 4-Ac-3,6-Me,nGlcNAc and 3,6-AC,-1,4,5-Me,N-acetyl-n-galactosaminitol. Periodate oxidation showed, per mol of oligosaccharide, 5.9 mol of periodate consumed (theory, 9.01, 0.4 mol of formaldehyde (theory, 0.0) and 3.7 mol of formic acid released (theory, 4.0). Glc analysis of the Smith-degraded oligosaccharide, after methanolysis and O-acetylation, showed destruction of Fuc and Gal. Periodate-resistant Gal and GlcNAc residues in a ratio of 2.3:1 (theory, 2.0) re- mains. N-acetyl threosaminitol was present in the methanolyzed and 0-acetylated portion but not in the portion 0-acetylated only, supporting the 3, 6-disubstitution of the N-acetyl-n-galactosaminitol. This compound shows strong B activity but is inac-

tive in the type XIV system (Fig. 2). The periodate oxidation data indicate that the compound probably contained some impur- ities; the amount isolated was insufficient for hplc. The tentative structure is given in Fig. 1. The 3, 6-disubstituted N-acetyl- n-galactosaminitol in compounds VII, VIII, IX and X could have the C-3 and C-6 branches reversed and still be consistent with the data. Galactose is assigned in linkage to C-3 by analogy with compounds II, III, IV and VI, leaving the GlcNAc on C-6. This assignment is consistent with the structures proposed for 3, 6-disubstituted N-acetyl-D-galactosaminitol containing oligosaccharides previously isolated from human Le” and HLe” cysts (11).

Figure 4 shows the ORD and CD curves for three oligosaccharides from non-B-active horse mucosae. In Table III are listed the values for [mltrOuph, LmL,, [m1300, [ml22o -[ml:soo, and the values of molecular ellipticity, [el,,,, at the extremum of the n -+ 7~* Cotton effect of the X-acetamido group (40). Table III also includes values for sev- eral other related compounds.

ORD and CD curves for RG0.87 show that maxima were not obtained, as noted previously for nGal/31+3nGalNAc (61, 2- acetamido sugar alcohols, including N- acetyl- D- galactosaminitol (41) and for ~Fucpl-+3~GlcNAc (42). Variations in ORD and CD maxima have also been seen (40). The [ml220 -[mlsoo value of -5272 may not be comparable to [mlz,o -[ml,3,, values for compounds showing a clear trough. The value for R,,0.87 is more negative by about 1400” than the value for N- acetyl-n-galactosaminitol (Table III and Ref. (41)). R,.1.04 shows a very strong Cot- ton effect at 220 nm, indicating a p-linkage from DG~cNAc to N-acetyl-D-galactosaminitol. The [ml22,, -Im&, value of -14,259” is considerably more negative than the sum of [mlzzo -[mLoo values for R (;0.87 (-5272”) and nGalfi4nGlcNAc (-2940”), being too negative by about 6000”. The tetrasaccharide R,,0.88 shows a Cotton effect at 220 m-n; a [mlzzo -[ml3oo value of -5577” is considerable more positive than the value for R,,1.04 (-14,259”). The sum of [mlzzo - [ml,oo values for R (;0.87 c-5272”), methyl a~GlcNAc (+ 1930”) and DGalpl+


4~GlcNAc (- 2940”) gives approximately -6300”, in good agreement with the ob- served value for R,0.88 of -5577".

The CD spectra are less informative. CD curves of R,0.88 and R,,1.04 are indistin- guishable. The [01,,, of R,,0.87 is signifi- cantly less than for either compound and about 3000” more negative than the value for N-acetyl-n-galactosaminitol (411, but since the extremum for each was not reached it is not possible to make any relia- ble quantitati.ve estimates.

DISCUSSION

A characteristic feature of oligosaccharides released by NaOH-NaBH, from horse gastric mucosal blood group glycoproteins is that about 1% or less of the reduced oligosaccharides are nondialyzable and, of those which are dialyzable, almost all are included in Bio-Gel P-2. Thus the upper size limit for at least 90% of the carbohydrate is probably in the molecular weight range of 1600-2000, corresponding approximately to 8-12 sugar residues. RIMsO. (X), a reduced octasaccharide with two B determinants was isolated and may represent complete expression of the biosynthetic potential of B-active horse mucosae; the smaller oligosaccharides R,,,1.15 (VIII) and RIM,1.18 (IX) may result from incomplete biosynthesis. Incompleteness is not meant to imply lack of function, as no role has yet been established even for the most complete blood group carbohydrate chains. Indeed, heterogeneity of the oligosaccharide moiety may prove an important functional characteristic of such glycoproteins with blood group activity. The recent demonstration of A and B activities in purified preparations of human maltase and sucrase associates blood group activity for the first time with a functioning protein molecule (43).

The oligosaccharides isolated comprise 5% or less of the original carbohydrate treated with base-borohydride. This is in large part due to losses in the many chromatographic steps and from adsorption to the ion-exchange resins used in desalting (9). While much less heterogeneous than materials derived in identical fashion from human ovarian cysts (16, 44). Bio-Gel P-2

fractionation and paper chromatography revealed a quite complex mixture with cer- tain oligosaccharides in predominant amounts; some of these have been characterized.

All compounds reported are pure by four or more criteria. Two minor fractions of oligosaccharides contain significant amounts of inert weight (footnote c, Table I), often a problem in the purification of less than 5 mg of material (11). The structures of R ,,0.69 (VII), R,,,1.15 (VIII) and R&.18 (IX) were not based on quantitation of N-acetyl-n-galactosaminitol and quantitative periodate oxidation because of the small amounts isolated. Each was pure by all criteria applied. Sufficient in- formation exists from calorimetric, methylation and immunochemical analyses to support the structures in Fig. 1.

nGalpl-+3N - acetyl - D- galactosaminitol (11, 13, 14,461 and nGalpl-+SnGalNAc (45) have been previously isolated from hog and human sources. LFucal-+2nGalpl-+3 N-acetyl-n-galactosaminitol (III) has also been isolated from hog (14, 46) and human sources (11). The seven remaining oligosaccharides have not been previously found. No B-active oligosaccharides were isolated from non-B-active materials. Only 4 mg of R,M150.65 (IV) were obtained; it consumed only 5.9 mol of periodate per mol of compound (theory, 9.0). No explanation can be offered for this value, but it is inconsistent with the release of 3.7 mol of formic acid (theory, 4.0) per mol of compound. The somewhat high value for formaldehyde released has been encountered previously for oligosaccharides containing 3, 6-disubstituted N-acetyl-n-galactosaminitol. Colori- metric data, methylation and Smith degradation analyses are consistent with the structure in Fig. 1.

The B-active tetrasaccharide R ,,0.65 (IV) bears a strong resemblance to two A-active oligosaccharides isolated from pig submaxillary mucin (46); both A-active oligosaccharides contain a terminal a-linked nonreducing nGalNAc in place of a-linked DGal (Fig. 1). One of the A-active oligosaccharides also has N-glycolylneuraminic acid linked a2 + 6 to N-acetyl-n-galactosaminitol. R,,0.65 is the most abundant oligo-

saccharide from either material and may be responsible for the high B activity of horse B substance. Oligosaccharides containing the larger determinant DGal(Yl-+ 3[LFuc(u1+2]DGal/3+3 or 4 DG~cNAc have been shown to be better inhibitors of B precipitation than smaller oligosaccharides. The contribution of the DG~cNAc to the determinant has been shown by inhibition of B anti-B precipitation with oligosaccharides isolated from mild acid-hydro- lyzed B substance (Pl fraction) from human ovarian cyst (15). In that study Beach R,,0.26 and Beach R,,0.44 (Fig. 3) were equally potent and were the best inhibitors. Oligosaccharides from human ovarian cyst blood group substances contain two kinds of chain sequences: type 1 DGalpld3DGlcNAc and type 2 DGalPl+ 4~GlcNAc (47). Beach RL0.26 and Beach RL0.44 represent the type 1 and type 2 monofucosyl B determinants, respectively. Less potent as inhibitors but roughly equal were two oligosaccharides, R,0.62 and R,O.52-0.59. R,0.62 is equivalent to Beach R,0.44 minus Fuc and R,0.52-0.59 is equivalent to Beach R,0.26 minus Fuc. Least active were oligosaccharides DGalal +3DGal and DGalal+3Dgalactitol.

The present study shows that substitu- tion of a 3-linkedN-acetyl-D-galactosamini- toi for the DG~cNAc produces a substantial reduction in B activity. Unfortunately the compound with the intact DGalNAc has not been isolated. However, Escherichia coli O,, lipopolysaccharide was as active in inhibition of hemagglutination as B substances from human ovarian cyst and horse gastric mucosae. It contains a determinant with the structure partially established as

~Fucnl ~Glcfll

J 1 ? 6

DGalal+SDGalp-+J or 4DGalNAcpl-+3DGalNAc-.

The decisive importance of quantitative inhibition of precipitation in elucidation of oligosaccharide structure is illustrated with R&.15 (VIII) and R iM51. 18 (IX) for which methylation data permitted two al- ternatives. Assavs showed R,,J.15 to be ” . I..y


type XIV active while R iM51. 18 was not. In the B system, R&.18 was more potent than R&.15 and comparable to B oligosaccharides containing DG~cNAc (Beach R,,0.26 and Beach R,0.44); RnJ.15 was equivalent to R ,>0.65 (IV) in inhibition of B precipitation over the range studied and therefore possessed a B determinant on the branch linked j31 + 3 to N-acetyl-D- galactosaminitol.

The trisaccharide RJ.04 (V) (2.3 mg) may represent a structure unique to horse blood group substances in that it contains ~GlcNAcpl -+ 3 to N-acetyl-D-galactosaminitol. In all mammalian blood group oligosaccharides characterized to date, the sugar in 1 -+ 3 linkage in N-acetyl-D- galactosaminitol, i.e., the DGalNAc in- volved in linkage to serine or threonine, has been DGal.

The tetrasaccharide RlA0.88 (VI) resem- bles oligosaccharides previously isolated in this laboratory from hog mucin A + H substance in having a terminal nonreducing a-linked DG~cNAc (5,6). R,0.88 has not been isolated from hog gastric mucosae but is analogous to the proposed linkage region of sulfated hog gastric mucosae glycoproteins (12). The occurrence in hog A + H substances from gastric mucosae of two or more DG~cNAc residues in sequence has been reported (49). While B- active fractions precipitated Con A as well as non-B-active (9,28), no oligosaccharides with terminal nonreducing a-linked DG~cNAc were isolated isolated from the former.

RL0.69 (VII), R,M51.15 (VIII), RIM51.18 (IX) RIMsO. (X1 all possess 3,6-disubstituted N-acetyl-D-galactosaminitol, a branching feature well documented oligosaccharides from NaOH-NaBH,-treated ovarian cyst precursor I, Lea and HLe” glycoproteins (10, 11, 13, 15) but has not been reported in oligosaccharides from hog blood group substances. Hog submaxillary mucin (46) treated with NaOH-NaBH, yielded the 3,gdisubstituted N-acetyl-D- galactosaminitol as

DGalPl -P 3?V-acetyl-wgalactosaminitol.

N-glycolyl neuraminic acid&

J 6


The core structure shared by horse and human materials is that for N-l RIP.44 (Fig. 3) (11) and various oligosaccharide chains synthesized upon this structure may contribute further to species specific- ity and heterogeneity of blood substances.

Horse-derived oligosaccharides differ from human mainly in the sizes of their largest oligosaccharide chains, the latter being longer (11) and having more highly branched structures leading to multiple determinants (LO). No oligosaccharides with either type 1. chains or structures involv- ing more than two sugars built on the DGal linked /31+ :3 to N-acetyl-n-galactosamini- to1 of the branched tetrasaccharide N-l R,0.44 (Fig. 3) isolated from human blood group substances were obtained. Acetoly- sis of hog sulfated gastric mucin A + H substance have been interpreted as indicating (50) the presence of a 3,6-disubstituted DGal as occurs in human cyst substance. The former, however, has two nGa1 residues while the latter has two DG~cNAc residues. Hog materials also have type 1 chains (7,50:1. The sizes of the largest oligosaccharide chains from hog gastric mucosal blood group-active substances are closer to 14-16 residues as in human ovarian cyst oligosaccharides than to 8-12 residues as in horse substances. Type 1 chains have been associated most often with large oligosaccharides and may even be more common in fractions containing larger oligosaccharides which have not been characterized (11). This could be related to the failure to isolate type 1 chains from horse materials although they have been found in hog and human blood group substances. Unlike horse mucosal blood group substances, those from human ovarian cyst treated with base-borohydride (11, 13, 16) yield substantial amounts of unsubsti- tuted N-acetyl-n-galactosaminitol. This may be related to the maximal chain sizes in the two species allowing smaller oligosaccharides on the peptide backbone more access to glycosyl transferases.

ORD and CD spectra of various mono- and oligosaccharides with 2-acetamido groups established that the Cotton effect in the 220 nm region was probably due to the n -+ r* transition of the acetamido

group (40). The nature of the glycosidic linkage, (Y or p, was the most important factor determining the size and sign of the Cotton effect (40-42). The contribution of other chromophores was minimized by sub- tracting the [rnlSoo value from the [mltrouph or, in the absence of a clear trough, from the [mlzzo value. The regularity of [ml22o --[tnls~ for compounds with the same glycosidic linkage, cu or p, or in oligosaccharides with the same substituent linkage 1 + 3, 1 + 4, or 1 + 6 permitted some general inferences about their contribution to the size and sign of the Cotton effect at 220 nm. ORD spectra of RG0.87 (II), R J.04 (V) and R L,0.88 (VI), not previously examined, are consistent with results of chemical, immunochemical and enzymatic analyses for the structures in Fig. 1.

The nature of the carbohydrate chains responsible for the B and other activities of blood group active substances from bovine stomach, last studied 23 years ago (31, would yield further insights into the nature of species differences among blood group active glycoproteins and to our over- all knowledge of glycoprotein structure.

ACKNOWLEDGMENTS

We thank Dr. Sherman Beychok for advice in interpretation of the ORD and CD data, Dr. Lajos Bandy for performing the infrared analysis ofR,6.88 and Dr. Luciana Rovis, Dr. Kenneth 0. Lloyd, and Dr. Miercio Pereira and Dr. Florence Maisonrouge McAuliffe for many helpful discussions.

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Documents

Immunochemical studies on blood groups: Structures and immunochemical properties of nine oligosaccharides from B-active and non-B-active blood group substances of horse gastric mucosae