- Email: [email protected]
[45] Snail (Helix pomatia) hemagglutinin
368
PURIFICATION OF CARBOHYDRATE-BINDING
PROTEINS
[45]
all properties tested, including amino acid composition, carbohydrate content, migration on acrylamide gel, and biological specificity, the agglutinins prepared by both methods appear to be identical. However, SBA prepared by method II is a mixture of all four agglutinins. The major SBA can be obtained free of the three minor components, by chromatography on DEAE-cellulose as described under method I.
[45]
Snail (Helix pomatia) H e m a g g l u t i n i n
By
STEN HAMMARSTROM
The albumin gland of the snail Helix pomatia (class Gastropoda, phylum Mollusca) contains a protein (Helix pomatia A hemagglutinin) which agglutinates human A erythrocytes but not human B or 0 erythrocytes. 1,2 The hemagglutinin is also found in the eggs but not in the hemolymph2 The albumin gland, which is part of the sexual apparatus, contains relatively large amounts of the protein (approximately 8% of total protein in the combined supernatants after PBS extraction and ultracentrifugation, see below). The hemagglutinin seems to be evenly distributed over the entire gland as visualized by indirect immunofluorescence staining with rabbit antisera against the purified hemagglutinin.3 Similar hemagglutinins have been detected in total extracts of snails from the species Helix hortensis 4 and Otala lactea. 5 These agglutinin-containing extracts and Helix pomatia A hemagglutinin seem to have approximately the same specificity as judged from their ability to agglutinate normal and enzyme treated erythrocytes of different vertebratesY Only Helix pomatia A hemagglutinin has been purified and studied in greater detail. Purification The hemagglutinin from Helix pomatia is easily purified by immunospecific adsorption to insoluble human or hog blood group A substance followed by elution with D-GalNAc2 The procedure is as follows: The albumin glands from about 200 snails (approximately 90 g wet weight) 10. Prokop, D. Schlesinger, and A. Rackwitz, Z. Immun. Forsch. 129, 402 (1965). 20. Prokop, G. Uhlenbruck, and W. KShler, Vox Sang. 14, 321 (1968). S. HammarstrSm, unpublished observations. ' O. Prokop, A. Rackwitz, and D. Schlesinger, J. Forensic Med. 12, 108 (1965). 5W. C. Boyd and R. Brown, Nature (London) 208, 593 (1965). 6S. Hammarstriim and E. A. Kabat, Biochemistry 8, 2696 (1969).
[45]
SNAIL HEMAGGLUTININ
369
are dissected out, homogenized in a blender, and extracted in the cold with 200 ml of 0.15 M phosphate-buffered saline, pH 7.2 (PBS). The extract is centrifuged at 12,000 rpm for 20 minutes at 4 °, and the precipitate is reextracted with the same volume of buffered saline. The combined supernatants are then spun in the ultracentrifuge at 40,000 rpm for 2.5 hours. Three fractions are obtained; a clear, light green supernatant, a light precipitate, and a heavy gelatinous precipitate. The supernatant contains by far the highest concentration of hemagglutinin and is used for further purification. The heavy precipitate consists essentially of galactan. The clear supernatant is then passed repeatedly through a column of carefully washed insoluble hog blood group A + H substance, mixed with Celite (1:1). Saturation is monitored by measuring the absorbance at 280 nm and the hemagglutinating activity of the solution before and after passage through the column. At saturation the column is washed extensively with PBS until the absorbance at 280 nm is below 0.050. Specific elution is effected by 0.005-0.015M D-GalNAc in PBS. The eluted material is concentrated by ultrafiltration and passed twice through a column of Biogel P-10 to remove free and bound n-GalNAc. Insoluble hog blood group A + H substance is prepared by copolymerization with the N-carboxyanhydride of L-leucinC as described by Kaplan and Kabat? The hemagglutinin binding capacity of polyleucyl hog blood group A + H substance is approximately 30% on a weight basis. 6 The crude extract contains in addition to Helix pomatia A hemagglutinin a second component6 (Fig. 1B), which also combines with hog blood group A + H substance. This protein, which does not agglutinate human A erythrocytes, can be removed by preeluting the column with 0.05 M D-Glc or by extensive washing with PBS. In a typical experiment~ 85.5% of the adsorbed hemagglutinin was recovered after elution with 0.005 M D-GalNAc. Very little additional nitrogen, however, was recovered by raising the D-GalNAc concentration to 0.05M (0.01%) or by elution with 2 M KSCN (0.7% of total eluted nitrogen). The latter fraction contained only nonhemagglutinin components, indicating that the hemagglutinin is completely eluted. The hemagglutinin can also be purified by adsorption to Sephadex G-200 and elution with D-GalNAc, D-Gal, or even D-Glc in the same manner as described for insoluble hog blood group A + H substance2 '1° H. Tsuyuki, H. von Kley, and M. A. Stahmann, J. Amer. Chem. Soc. 78, 764 (1956). s M. E. Kaplan and E. A. Kabat, J. Exp. Med. 123, 1061 (1966). O. Kiihnemund and W. KShler, Experientia 25, 1137 (1969). ,o I. Ishiyama and G. Uhlenbruck, Z. Klin. Chem. Klin. Biochem. 9, Heft 5 (1971).
370
PURIFICATION OF CARBOHYDRATE-BINDING PROTEINS
~.~
[45]
o.
6
z
4~
t28
~ u
E<
ooE ~
2
v
0
I'o
v
2' 0
v
' 30
4'0
' 5O
6 'o
~o
"-°
/.zg antigen added
Fla. 1. Ultraeentrifugal, immunoeleetrophoretic, and precipitin pattern of immunosorbent purified Helix pomatia A hemagglutinin. (A) Schlieren pattern of purified snail hemagglutinin [fraction II of S. HammarstrSm and E. A. Kabat, Biochemistry 8, 2696 (1969)] (1.30 mg of N per milliliter in 0.9% NaC1). The photomicrograph was taken after 144 minutes at 50740 rpm. (B) Immunoelectrophoretic analysis of purified snail hemagglutinin, fraction II, 1.30 mg of N per milliliter (center well), and of crude extract of albumin gland (upper and lower wells); rabbit antiserum against crude extract of albumin gland (upper trough) and hog blood group A + H substance (lower trough) were added to develop reactions. (C) Precipitation of p.urifled snail hemagglutinin fraction II, 6A7 gg of N per tube, by human blood group A substance (cyst MSM) ((~--O), The total volume was 200 #l. Hemagglutination titer of supernatants against human Al-erythrocytes (0--{}). I t is likely t h a t nonreducing a-linked D-Glc end groups in dextran are responsible for the binding of the hemagglutinin. This is a v e r y w e a k interaction since D-Glc can displace the hemagglutinin from the Sephadex column. Precipitin inhibition studies furthermore show t h a t D-Glc or Me-a-D-Glc are v e r y poor inhibitors as c o m p a r e d with D-GalNAc (see below). T h e a b u n d a n c e of D-Glc end groups in dextran and the high v a l e n c y of the hemagglutinin seem, however, to compensate for the w e a k -
[45]
SNAIL HEMAGGLUTININ
371
hess of the interaction. A similar situation has been described for certain group C antistreptoeoccal antibodies? ~ Purity Immunosorbent purified Helix pomatia A hemagglutinin is homogeneous on gel filtration on Biogel P-150 and P-300 and gives only one symmetrical peak in the analytical ultracentrifuge 6 (Fig. 1A). It gives only one line in immunoelectrophoresis even when tested at very high protein concentration with antiserum to the crude extract6 (Fig. 1B). With this antiserum at least 15 components were detected in the crude extract (Fig. 1B). It is furthermore completely precipitated by human blood group A substance 6 (Fig. 1C). Polyacrylamide gel electrophoresis at alkaline pH revealed, however, a certain degree of heterogeneity. Although approximately 90% of the material eluted with 0.005-0.015 M n-GalNAc appeared as one band, three additional bands with closely similar electrophoretic mobilities were seen. The relative amount of these bands was different in fractions eluted earlier from the immunosorbent column than in fractions eluted later. In the last fraction (1.3% of total eluted hemagglutinin) three bands (including the major band) of approximate equal density were obtained. Since this fraction, as well as the earlier fractions, were completely precipitated by human blood group A substance B it follows that all bands must represent the Helix pomatia A hemagglutinin. Polyacrylamide gel electrophoresis at different gel concentrations showed furthermore that these bands differed in charge but not in size2 This may either reflect a heterogeneity of the starting material--a pool of albumin glands from several hundred snails was used--or may be due to secondary chemical changes. Chemical and Physicochemical Properties The sO0,wvalue for the hemagglutinin was determined to 5.3 s (0.9% NaC1). From this, the intrinsic viscosity, and the partial specific volume, a molecular weight of 1.0 × 105 was calculated2 Further ultracentrifugation studies, 12 using the meniscus depletion sedimentation equilibrium method, showed that this value was too high. A mean molecular weight of 79,000 was obtained. TM Amino acid analysis shows that the hemagglutinin contains approximately 18 moles of half-cystine and 10 moles of methionine per molecu11T. J. Kindt, C. W. Todd, K. Eichmann and R. M. Krause, J. Exp. Med. 131, 343 (1970). ~S. HammarstrSm, A. West55, and I. BjSrk, Scand. J. Immunol. (1972) in press.
372
PURIFICATION OF CARBOHYDRATE-BINDING
PROTEINS
[45]
lar weight of 79,000. 6 Carbohydrate analysis revealed the presence of D-galactose (4.0%) and D-mannose (3.3%)2 Trace amounts of hexosamine were also detected. It is, however, likely that the latter represents residual D-GalNAc not completely removed by dialysis and gel filtration. Subunit Structure Direct alkylation of the hemagglutinin at pH 8.6 either in Tris buffer alone or in 6 M guanidine.HC1 showed that free sulfhydryl groups were absent 12 (Table I). Reduction with excess dithiothreitol (DTT) in 6 M guanidine.HC1 lead, on subsequent alkylation, to the incorporation of approximately 18 moles of [14C]-acetate per molecular weight of 79,00012 (Table I). Thus under these conditions the hemagglutinin was completely reduced. Gel filtration of the alkylated material on Sephadex G-100 in 6 M guanidine.HC1 gave the elution pattern shown in Fig. 2A. One symmetrical included peak, in addition to aggregated material in the exclusion volume, was obtained. 12 Under these conditions the unreduced hemagglutinin is included22 The mean molecular weight of the included component (Helix pomatia A hemagglutinin subunit) was 13,000 as determined from a calibration curve for the same column, using completely reduced and alkylated proteins of known molecular weights ~ (Fig. 2B). The amino acid composition showed that the hemagglutinin per molecular weight 13,000 contains 12 moles of lysine and arginine and 3 moles of half cystine. Thus, if the protein consists of identical subunits, 13 peptides with maximally 3 containing cysteine would be expected on tryptic digestion. In fact, 13-14 maior peptides (2 acid, 6 neutral, and 5 or 6 basic peptides) were found after combined electrophoretic and chromatographic analysis 13 of tryptic digest, prepared from reduced and ~4C-carboxymethylated or performic acid oxidized hemagglutinin. 12 Three of these peptides were radioactive. One of them (acid peptide) gave however a more intense radioactive spot. 12 These data suggest that the hemagglutinin consists of only one type of subunit with an approximate molecular weight of 13,000. Reduction in the absence of unfolding agents followed by alkylation revealed that disulfide bonds were not cleaved ~2 (Table I). Treatment of the hemagglutinin with 6 M guanidine.HCl at pH 4.0 or with 6 M guanidine.HC1 alone for 15 minutes up to 2 days gave one component with a molecular weight of 26,000-31,000 both on gel filtration in 6 M guanidine.HC1 + 0.1 M D-GNAc and on ultracentrifugation (meniscus depletion sedimentation equilibrium method).1_- Moreover the :a H. JSrnvall, Eur. J. Biochem. 16, 41 (1970).
[45]
SNAIL HEMAGGLUTININ
373
X
0
v
>
V
• --
~
~
~.~
~
• .-
0
m
~
o
~
~
~t.-d
0 Z 0
0
v
= N
I ~
.
374
PURIFICATION OF CARBOHYDRATE-BINDING PROTEINS
A
[45]
B
A280
0/-~0 Ribonuclease
0.30
..... ~ s o z y m e l
1600
Q20
1.60
i
"~Trypsin
i 0.10
1.20 |
=
20
.
.
.
.
.
•
"
~
/-,0 60 80 FRACTION
|
100
0
120
-~ConA
I
~
Pepsin
i I
i
i
L
10 20 30 40 _503 MOLECULARWEIGHT X 10
FIG. 2. Gel filtration profile of completely reduced and [14C]alkylated Helix pomatia A hemagglutinin on Sephadex G-100 column in 6 M guanidine'HC1 (A) and calibration curve for the same column (B). (A) The solid line denotes absorbance at 280 nm, and the dashed line denotes radioactivity. The column was 2.5 × 40 cm. The exclusion volume (Vo) is indicated in the figure. (B) Completely reduced and alkylated proteins were used for calibrations. The dashed lines denote the V~/Vo value and the molecular weight for the major component of Fig. 2A. Con A, concanavalin A.
hemagglutinin did not dissociate further when treated with 7 M guanidine.HC1 containing 1 M propionic acid as determined by gel filtration experiments in this medium.12 Control experiments demonstrated that the hemagglutinin was not reduced under these conditions (0.2 mole [14C]acetate per molecular weight 79,000). Conditions were also found that gave rise to partial reduction of the hemagglutinin (e.g., reduction with excess DTT in 8 M u r e a _ 0.1 M D-GNAc or in 6 M guanidine-HC1 containing 0.1-0.2M D-GalNAc or D-GNAc). 3-4 disulfide bonds were cleaved under these conditions12 (Table I). Gel filtration in dissociating agents in the presence of D-GNAc gave only one symmetrical component with a molecular weight of 12,50016,700 (determined from standard curves for reduced and unfolded proteins or non-reduced but unfolded proteins respectively). This component furthermore contained an active carbohydrate binding site. 12 Taken together these data suggest that the hemagglutinin is made up of six identical or closely similar polypeptide chains, each containing one intrachain disulfide bond and a carbohydrate binding site. The subunits are arranged in pairs in which they are linked together by a single disulfide bond. Native hemagglutinin is formed by the interaction of non-covalent forces between three dimers.
[45]
SNAIL HEMAGGLUTININ
375
Homogeneity of Carbohydrate BLnding Site A blood group A active pentasaccharide, ARL 0.52, a-D-GalNAc (i--> 3) - [q-L-Fuc- (i --> 2) ] -fl-D-Gal- (I ---> 4) -fl-D-GNAc- (I ~ 6) -3-hexenetetrol (s) labeled with tritium in the hexenetetrol residue I' was used in equilibrium dialysis experiments to investigate the binding properties of Helix pomatia A hemagglutinin. This structure constitutes the determinant that is responsible for precipitation of blood group A substance by the hemagglutinin (see below). Figure 3A demonstrates a linear relationship when the binding data 1~ are plotted according to Scatchard. 16 A heterogeneity index, a, of 1.04 was obtained when the binding data were plotted according to Sips distribution 17,18 (Fig. 3B). Thus, within the experimental error the binding sites are homogeneous. On the basis of a molecular weight of 1.0 × 105 for the hemagglutinin, the equilibrium dialysis data indicate that the hemagglutinin contains one combining site/molecular weight of 16,000-17,000.1~ This value is in reasonable agreement with the molecular weight of the subunit.
B
A
50 25 "T
%
2O
0
15 o~
-I
I0
o
I
60
I
L
L
t
2
3
tog c (pM)
Fro. 3. Equilibrium dialysis experiments with purified Helix pomatia A hemagglutinin and tritium-labeled A active pentasaccharide [3H]AR~ 0.52. The data are plotted both according to Scatehard (A) and according to Sips (B). Approximately 12 mg of snail protein was used for each determination. 1, C. Moreno and E. A. Kabat, J. Exp. Med. 129, 871 (1969). ~S. tIammarstrlim and E. A. Kabat, Biochemistry 10, 1684 (1971). 16G. Scatchard, Ann. N.Y. Acad. Sci. 51, 660 (1949). 1TR. Sips, J. Chem. Phys. 16, 490 (1948). ~SF. Karush, Advan. Immunol. 2, 1 (1962).
376
PURIFICATION OF CARBOHYDRATE-BINDING PROTEINS
[45]
Further evidence for homogeneity of binding sites has been obtained by equilibrium dialysis displacement experiments 15 (Fig. 4). In this experiment the capacities of the cross reactive haptens Me-a-D-GalNAc, Me-a-D-GNAc, and Et-fl-D-GNAc to displace the labeled A active pentasaccharide, [3H]ARL0.52, from the binding site were investigated. Linear displacement curves essentially parallel with the self-displacement curve, ARL0.52, were found in the plot. Thus over the range tested (from 10 to 70-80% displacement) there is no substantial heterogeneity of association constants for the binding of the displacing haptens. Specificity of Carbohydrate Binding Site The specificity of the binding site of Helix pomatia A hemagglutinin has been investigated by the following procedures. Equilibrium Dialysis and Displacement. The intrinsic association constant (Ko) for the interaction of the hemagglutinin site with Me-a-DGalNAc or the blood group A active pentasaccharide is 5.0 × 103 1/mole at 25 ° and pH 7.215 (Table II; Figs. 3 and 4). These compounds are the best inhibitors of precipitation studied so far. The Ko values for Me-a-D'5 (D 0 I,¢ O4
d
II0 O
I00
•
n
9O .J
E
80
,-~
70
Z
~ 6o u. 0 z o ~ er
~
50 40 30 2O
Z
uJ U Z 0 ¢J
IO I
-5 I0
I
I0
l
-4
I0
I
-3
I0
-2
FREE CONCENTRATION OF COMPETITOR(M)
FIG. 4. Equilibrium dialysis displacement experiments with unlabeled ligands. Approximately 12 mg of hemagglutinin and a concentration of ['H]ARL 0.52 which gives 110 × 1 0 - ' M bound radioactive h a p t e n were employed for each determination. Displacing haptens were ARL0.52 (O----O), self displacement curve; Me-a-D-GalNAc ( D - - D ) , M e - a - , - G N A c ( A - - A ) , and Et-fl-D-GNAe ( O - - O ) . The dashed horizontal line signifies the concentration of bound radioactive hapten in the absence of competitor.
[451
SNAIL HEMAGGLUTININ
377
GNAc and Et-fl-D-GNAc were approximately 4.5 and 27 times lower than for Me-a-D-GalNAc, respectively 1~ (Table I I ; Fig. 4). Although the intrinsic association constants for the best inhibitors are relatively low, they are of the same order of magnitude as for other carbohydrateanticarbohydrate systems. 1~,19,2° Inhibition of Precipitation. The most complete data on the specificity of the hemagglutinin site have been obtained by inhibition of precipitation using either human blood group A substance or Salmonella typhimurium SH 180 lipopolysaccharide (LPS) as test antigens2 ,1~,21 These two systems were used since they differ markedly in sensitivity. With the same amount of hemagglutinin, approximately 1000 nmoles of added D-GalNAc is needed for 50% inhibition in the former system whereas 25 nmoles of added v-GalNAc is required for the same degree of inhibition in the latter. The reason for this difference is that the hemagglutinin reacts with a-linked D-GalNAc in blood group A substance, ~ whereas the structure, a-D-Gal (1--)6)-D-Glc..., is "immunodominant" in S. typhimurium SH 180 LPS. 21 Table III shows the concentrations of various monosaccharides, methylglycosides, and oligosaccharides needed for 50% inhibition. The data are based on complete inhibition curves. In summary the results show: (1) Me-a-D-GalNAc is the best inhibitor of all compounds investigated so far. (2) In the series of a-DGalNAc containing oligosaccharides and derivatives, only the terminal nonreducing D-GalNAc residue seems to bind significantly to the site. This may indicate that the combining site is relatively small. (3) Me-a-DGalNAc is only about 4 times more active than Me-a-D-GNAc, indicatTABLE II INTRINSIC ASSOCIATION CONSTANTS (Ko) AND STANDARD FREE-ENERGY CHANGE (--AF °) FOR THE INTERACTION OF Helix pomatia A HEMAGGLUTININ WITH VARIOUS HAPTENS
AT p H
7.3 A N D 25.0 + 0.i °
Hapten
Ko X 10-a (M-1)
--AF ° (kcal/mole)
ARL 0.52a Me-a-D-GalNAc Me-a-D-GNAc Et-f~-D-GNAc
5.0 5.0 1.1 0.18
5.04 5.04 4.14 3.08
a See text. ~gL. L. So and I. J. Goldstein, J. Biol. Chem. 243, 2003 (1968). soM. Katz and A. M. Pappenheimer, Jr., J. Immunol. 103, 401 (1969). ~1S. HammarstrSm, A. A. Lindberg, and E. S. Robertson, Eur. J. Biochem. 255 274 (1972).
378
PURIFICATION OF CARBOHYDRATE-BINDING PROTEINS
[45]
T A B L E III CONCENTRATION OF MONOSACCHARIDES~ METHYLGLYCOSIDES~ AND OLIGOSACCHARIDES N E E D E D FOR 5 0 ~
I N H I B I T I O N OF P R E C I P I T A T I O N OF P U R I F I E D
Helix pomatia A
HEMAGGLUTININ WITH DIFFERENT CARBOHYDRATE ANTIGENSa Micromoles
for 50%
inhibition
of precipitation
of hemagglutinin With Inhibitor Me-a-D-GalNAc
blood
With
germfree
group A
rat colon
substance
antigen b
0.76
With
S. typhimurium SH
180 LPS
--
--
--
--
0.96
--
--
ART.0.52"
2.00
--
--
Ph-a-v-GalNAc
1.65o
--
--
o-{-p-NO2Ph-a-D-GalNAc
5.37g
--
a-D-GalNAc-(1
--* 3 ) - D - G a l
a-v-GalNAc,-(1
--~ 3 ) - ~ - v - G a l -
>0.14
(1 --* 3 ) - D - G N A c
Et-~-D-GalNAc ~ p-NO2Ph-B-D-GalNAc
~
> 3.60
--
--
> 0.99g
--
--
D-GaINAc d
1.65
D-GalNH2 •
> 9.73
v-GalNAc-oV
> 11.6
0.130
0.024
--
--
--
--
2--O-Ac-Me-a-D-Gal
--
0.050
--
2-O-Ac-Me-~-v-Gal
--
0.098
--
Me-a-D-GNAt
4.00
Et-~-D-GNAc D-GNAt
10.0 9.00
D-GNH~e
> 18.8
D-GNAc-ol /
> 11.6g
2-O-Ac-Me-a-v-GNAc 2-O-Ac-Me-~-D-GNAc Me-a-D-Gal a - D - G a l - ( 1 ---* 6 ) - D - G l c Me-~-D-Gal
--0.52 ---
0.070 -0.150 ---
--
1.60
--
--
3.50
--
> 10.9 -> 10.7
-> 5.40
7.00 3.20.
--
--
~ - D - G a l - ( 1 --, 3 ) - D - G a l N A c
>0.59
--
--
~ - D - G a l - ( 1 --~ 3 ) - D - G N A c
> 1.95
--
--
~ - D - G a I - ( 1 --~ 4 ) - D - G N A c
>2.09
--
~ - D - G a l - ( 1 --* 6 ) - D - G N A c D-Gal
> 1.31 >15.8
->2.40
--10.5
Me-~-D-Glc
>8.50
--
Me-~-D-Glc
> 17.9
--
24.1 --
n-Glc
> 18.4
--
40
Me-a-D-Man
> 39.0
--
55
D-Man
> 22.3
--
80 --
Me-a-D-ManNAc
> 5.32
--
Me-B-D-ManNAc
> 4.08
--
--
D-ManNAc
> 11.3
--
--
L-Fuc
> 15.8
--
> 150
[45]
SNAIL HEMAGGLUTININ
379
TABLE III (Continued) Micromoles for 50% inhibition of precipitation of hemagglutinin
Inhibitor #-L-Fuc-(1 --+ 3)-D-GNAc D-Xyl D-Ara S. typhimurium TV 160 core fragment S. typhimurium SH 180 core fragment
With blood With germfree With group A rat colon S. typhimurium substance antigenb SH 180 LPS > 1.79 -----
------
-95 > 150 0.70 4.50
Precipitation was performed in a total volume of 200 ~1 except for S. typhimurium SH 180 LPS where 400 ~1 was used. The following amounts of hemagglutinin and carbohydrate antigens (= equivalence point) were used: 12.8 I~g of human blood group A substance (cyst MSM) + 5.2 pg N of hemagglutinin; 11.8 I~g of germfree rat colon antigen -{- 3.5 ~g N of hemagglutinin; 88.7 ~g of S. typhimurium SH 180 LPS + 3.4 ~g N of hemagglutinin. b Rat colon antigen was obtained by phenol-water extraction of feces from germfree rats. The preparation was purified by ethanol precipitation (55-65% ethanol fraction) and treated with neuraminidase. c See text. d The capacities of these three compounds to give 50% inhibition of precipitation of human blood group H substance, first stage periodate oxidation and Smith degradation (cyst JS; 14.9 ~g) with the hemagglutinin (3.9 ~g N) were also studied [S. HammarstrSm and E. A. Kabat, Biochemistry 10, 1684 (1971)]. The concentrations needed were 0.031, 0.072, and 0.010 ~mole/200 ul for Et-f~-D-GalNAc, p-NO2Ph-f~-n-GalNAc, and D-GaINAc, respectively. D-GalNH2, 2-amino-2-deoxyoD-galactose; D-GNH2, 2-amino-2-deoxy-D-glucose. J D-GalNAc-ol, 2-acetamido-2-deoxy-])-galactitol, D-GNAc-ol, 2-acetamido-2-deoxyD-glucositol. a These values are calculated from inhibition data obtained with 9.4 j~g of human blood group A substance (cyst MSM) and 3.9 I~g N of hemagglutinin. With this amount of hemagglutinin 0.92 ~moles of D-GalNAc was needed for 50% inhibition as compared to 1.65 #moles in the system shown in the table. ing t h a t the steric o r i e n t a t i o n of the h y d r o x y l group on C - 4 is of lesser i m p o r t a n c e . (4) T h e presence of a n e q u a t o r i a l l y oriented N - a c e t y l or O - a c e t y l group on C - 2 is essential for strong b i n d i n g to t h e site. (5) All a - g l y c o s i d i c a l l y linked c o m p o u n d s b i n d more s t r o n g l y to the site t h a n the corresponding f l - l i n k e d derivatives. (6) Sugars t h a t a r e i n a n y w a y s t r u c t u r a l l y r e l a t e d to D-GalNAc can be shown to i n t e r a c t w i t h t h e h e m a g g l u t i n i n site p r o v i d e d the a s s a y s y s t e m is m a d e sufficiently sensitive. Direct Precipitation. H u m a n blood group A substance, desialized ovine s u b m a x i l l a r y m u e i n , group C streptococcal polysaccharide, hog blood
380
[45]
PURIFICATION OF CARBOHYDRATE-BINDING PROTEINS
group A + H substances, and purified intestinal mucin from germfree rat~ are precipitated by the hemagglutinin. 3,6,15 In these macromolecules, multiple nonreducing a-linked D-GalNAc end groups are exclusively or predominantly responsible for the interaction with the hemagglutinin site. For human blood group A substance this has been rigorously shown by means of an N-deacetylase from Clostridium tertium2 This enzyme specifically removes the N-acetyl group on nonreducing D-GalNAe of the blood group A determinant. 22 Enzyme treatment reduced the precipitating ability of blood group A substance to approximately one-tenth that of the original preparation e (Fig. 5A). Re-N-acetylation restored its precipitating ability completely e (Fig. 5A). The hemagglutinin also precipitates teichoic acids from Staphylococcus aureus containing a-linked D-GNAc nonreducing end groups, but not those with fl-linked nonreducing D-GNAc 6 (Fig. 5B). The content of a-D-GNAc containing teichoic acids could be determined quantitatively in strains that have a mixture of both by using the 3528 strain as reference (this strain contains teichoic acid with a-linked D-GNAc nonreducing end groups exclusively). The hemagglutinin did not precipitate group A streptococcal poly-
5,0
A
B
4,0 ~3.0 z~2~0 to x a
0
i
=
10
o
.
20
.
.
.
.
=
L
=
,
30 40 0 4 8 pg ANTIGEN ADDED
=
12
,
o
,
O
o- - ~ p ~ - -
16 20 24 28 4252
FIG. 5. Precipitation studies on hemagglutinin of fraction II of S. HammarstrSm and E. A. Kabat [Biochemistry 8, 2696 (1969)]. (A) Precipitation of purified Helix pomatla A hemagglutinin, 3~3 #g of N of fraction II, by human blood group A substance prior to (O) and after (O) treatment with blood group A N-deacetylating enzyme and of enzyme-treated and re-N-acetylated (A) A substance; total volume 200 ~l. (B) Precipitation of teichoic acids from different strains of Staphylococcus aureus (A, strain 3528; m, NYH-6; O, Copenhagen; ×, A1) and of S. albus (Prengel) (O) with purified snail hemagglutinin; 3.83 #g of N of fraction II, total volume of 200 #1. = D. M. Marcus, E. A. Kabat, and G. Schiffman, Biochemistry 3, 437 (1964).
[45]
SNAIL HEMAGGLUTININ
381
saccharide. 1~ In this polysaccharide D-GNAc is fl-linked to the rhamnosyl- (1--> 3)-rhamnose backbone. From these studies it would appear that macromolecules with sterically accessible nonreducing a-linked D-GNAc end groups, but not with fl-linked D-GNAc end groups, are precipitated by the hemagglutinin. The hemagglutinin also precipitates blood group substances of B, H, Le a, or precursor specificity,e The amount needed to precipitate a given amount of hemagglutinin varied within and between serotypes but was in all cases higher than that needed for blood group A substance. 6 Human blood group H substance also precipitates with the hemagglutinin after periodate oxidation and Smith degradation. The material was actually more active than untreated H-substanceY 3 A second cycle of oxidation and degradation (2nd periodate step) decreased the activity markedly. However, some activity was still left.23 The group(s) responsible for precipitation in blood group substances other than A is (are) not known. A possible candidate is a-D-GalNAc linked directly on serine or threonine in the peptide backboneY 4 In light of the data obtained with LPS from Salmonella typhimurium rough mutants (see below) other interpretations are however possible. Recent studies 21 have shown that lipopolysaccharides from certain rough mutants of S. typhimurium are precipitated by the hemagglutinin. While the LPS from the parent smooth strain was inactive, LPS from mutants of chemotype Ra and Rb and to some extent also chemotype SR were precipitated by the hemagglutinin. LPS from mutants of chemotypes Rc, Rd, Re as well as isolated lipid A were inactive. The reaction was immunologically specific and could also be demonstrated by independent techniques. Lipopolysaccharide core fragments prepared by weak acid hydrolysis and purified by gel filtration from the Rb mutant TV160 and the Ra mutant SH 180 both inhibited precipitation between the hemagglutinin and SH 180 LPS (Table III). The inhibition powers of these fragments were comparable to melibiose (a-D-Gal-(1 --->6)-D-Glc). The precipitating capacity of TV 160 lipopolysaccharide was abolished after treatment with the enzyme D-galactose oxidase. It was suggested that the structural element, a-D-Gal-(1 --> 6)-l)-Glc . . , present in all LPS from active mutants is the "immunodominant" structure responsible for precipitation. This finding is rather unexpected considering the poor inhibition obtained with Me-a-D-Gal or melibiose. However, in a situation where both reagents are polyvalent even very weak interactions may be sufficient for precipitation. ~' S. HammarstrSm and E. A. Kabat, unpublished observations. K. O. Lloyd and E. A. Kabat, Proc. Nat. Acad. Sci. U~g. 61, 1470 (1968).
382
PURIFICATION OF CARBOHYDRATE-BINDING PROTEINS
[45]
Utility Helix pomatia A hemagglutinin m a y be a useful tool for the detection of carbohydrate end groups on macromolecules and on cell surfaces. I t is already used routinely in blood group serology as a specific agglutinin for h u m a n A erythrocytesY 5 Its absolute specificity for h u m a n A erythrocytes as compared to B- and O-erythrocytes can be shown by tracelabeling it with 125iodine3 (Fig. 6). I t has furthermore been shown to be useful in group determination of fl-hemolytic streptococci. Thus group C and H streptococci are specifically agglutinated by the hemagglutinin. 2e Likewise Escherichia coli 026, 086, 0111, and 0126 are agglutinated b y the crude snail extract. 27 However, when used as a tool for the detection of particular carbohydrate end groups, mere demonstration of an interaction with an unknown macromolecule or cell is obviously not sufficient. C o m p l e m e n t a r y analysis is in most cases essential for identification of the end group. I n such analysis inhibition with haptens m a y be performed in order to compare the relative strengths of the reaction with an already established system involving the same anticarbohydrate reagent. The unknown macromolecule m a y furthermore be chemically or enzymatically treated to remove or modify the end group in question. The relatively broad
16
f
12 X
E OL
o-- AI
8
0
.~M
4 o
:
~.--,
o
2
e,
4 pg
o-GaI.NAc ,
6
~-B and 0
,.,
8
,
10
,-*,
,
12
[125I]HEEMAGGt.UTININ
Fie.. 6. Binding of radioactive Helix pomrltia A hemagglutinin to human A-, B-, and O-erythrocytes. Approximately 40 × 10~ cells were incubated with different amounts of 1-'SI-labeledhemagglutinin (specific activity = 2700 cpm/~g) in a volume of 2.0 ml. The cells were washed two times with PBS, and cell-bound radioactivity was determined by y-counting. 25C. HSgman, personal communications. '~ W. KShler and O. Prokop, Z. Immunol. Forsch. 133, 50 (1967). 27G. Uhlenbruck, 0. Prokop, and W. Haferland, Zentralbl. Bakteriol. I Orig. 199, 271 (1966).
[46]
EEL ANTI-H(O) GLOBULIN
383
specificity of the hemagglutinin tends to limit its usefulness in this respect. More useful may be the possibility to separate polysaccharides, glycoproteins, oligosaccharides or even cells with the aid of the hemagglutinin, made insoluble through polyeondensation or otherwise.
[46]
Eel S e r u m A n t i - H u m a n B l o o d - G r o u p H ( O ) P r o t e i n 1 B y PARIMAL R . DESAI a n d GEORG F. SPRINGER
Eel anti-human blood-group H(O) protein, a potent hemagglutinin of human blood-group 0 erythrocytes,2 is a 7 S globulin?,4 Originally it was thought that the combining sites of the eel antibody for bloodgroup H(O) specific structures are complementary to a-L-fucopyranose (6-deoxy-a-L-galactopyranose),~,6 but we found that monosaccharides with both L- and D-galactose configuration are complementary to these sites and function as inhibitory haptens provided they possess at least one methyl group on either C-3 or C-5. 7-1° Numerous other hexoses, pentoses and their derivatives are inactive. 7 The eel anti H(O) protein specifically precipitates not only with blood-group H(O) active macromolecules of human, animal, and plant origin, but surprisingly also with 3-O-methyl-D-fucose (D-digitalose) and 3-O-methyl-D-galactose.8,9,11 The minimum combining structure which shows inhibitory activity with the eel anti-H(O) antibody is smaller than a monosaccharide. It consists of a methyl substituent attached equatorially to a pyranose ring, an ether oxygen adjoining this methyl group, and an axial, oxygencarrying substituent cis to the methyl group on a contiguous C atom2 ,1° ' This investigation was supported by National Science Foundation Grant GB-8378. The Department is maintained by the Susan Rebecca Stone Fund for Immunochemistry Research. " S. Miyazaki, Nagasaki Idai Hoiqaku Gyoho 2, 542 (1930). A. Bezkorovainy, G. F. Springer, and P. R. Desai, Biochemistry 10, 3761 (1971). 4 G. F. Springer and P. R. Desai, Vox Sang. 18, 551 (1970). W. M. Watkins and W. T. J. Morgan, Nature (London) 169, 825 (1952). e R. Kuhn and H. G. Osman, Hoppe-Seyler's Z. Physiol. Chem. 303, 1 (1956). G. F. Springer and P. Williamson, Biochem. J. 85, 282 (1962). 8 G. F. Springer, P. R. Desai, and B. Kolecki, Biochemistry 3, 1076 (1964). 9 G. F. Springer, T. Takahashi, P. R. Desai, and B. J. Kolecki, Biochemistry 4, 2099
(1965). lop. R. Desai and G. F. Springer, Proc. lOth Congr. Int. Soc. Blood Trans]us., Stockholm, p. 500 (1965). 1~G. F. Springer and P. R. Desai, Biochemistry 10, 3749 (1971).
PURIFICATION OF CARBOHYDRATE-BINDING
PROTEINS
[45]
all properties tested, including amino acid composition, carbohydrate content, migration on acrylamide gel, and biological specificity, the agglutinins prepared by both methods appear to be identical. However, SBA prepared by method II is a mixture of all four agglutinins. The major SBA can be obtained free of the three minor components, by chromatography on DEAE-cellulose as described under method I.
[45]
Snail (Helix pomatia) H e m a g g l u t i n i n
By
STEN HAMMARSTROM
The albumin gland of the snail Helix pomatia (class Gastropoda, phylum Mollusca) contains a protein (Helix pomatia A hemagglutinin) which agglutinates human A erythrocytes but not human B or 0 erythrocytes. 1,2 The hemagglutinin is also found in the eggs but not in the hemolymph2 The albumin gland, which is part of the sexual apparatus, contains relatively large amounts of the protein (approximately 8% of total protein in the combined supernatants after PBS extraction and ultracentrifugation, see below). The hemagglutinin seems to be evenly distributed over the entire gland as visualized by indirect immunofluorescence staining with rabbit antisera against the purified hemagglutinin.3 Similar hemagglutinins have been detected in total extracts of snails from the species Helix hortensis 4 and Otala lactea. 5 These agglutinin-containing extracts and Helix pomatia A hemagglutinin seem to have approximately the same specificity as judged from their ability to agglutinate normal and enzyme treated erythrocytes of different vertebratesY Only Helix pomatia A hemagglutinin has been purified and studied in greater detail. Purification The hemagglutinin from Helix pomatia is easily purified by immunospecific adsorption to insoluble human or hog blood group A substance followed by elution with D-GalNAc2 The procedure is as follows: The albumin glands from about 200 snails (approximately 90 g wet weight) 10. Prokop, D. Schlesinger, and A. Rackwitz, Z. Immun. Forsch. 129, 402 (1965). 20. Prokop, G. Uhlenbruck, and W. KShler, Vox Sang. 14, 321 (1968). S. HammarstrSm, unpublished observations. ' O. Prokop, A. Rackwitz, and D. Schlesinger, J. Forensic Med. 12, 108 (1965). 5W. C. Boyd and R. Brown, Nature (London) 208, 593 (1965). 6S. Hammarstriim and E. A. Kabat, Biochemistry 8, 2696 (1969).
[45]
SNAIL HEMAGGLUTININ
369
are dissected out, homogenized in a blender, and extracted in the cold with 200 ml of 0.15 M phosphate-buffered saline, pH 7.2 (PBS). The extract is centrifuged at 12,000 rpm for 20 minutes at 4 °, and the precipitate is reextracted with the same volume of buffered saline. The combined supernatants are then spun in the ultracentrifuge at 40,000 rpm for 2.5 hours. Three fractions are obtained; a clear, light green supernatant, a light precipitate, and a heavy gelatinous precipitate. The supernatant contains by far the highest concentration of hemagglutinin and is used for further purification. The heavy precipitate consists essentially of galactan. The clear supernatant is then passed repeatedly through a column of carefully washed insoluble hog blood group A + H substance, mixed with Celite (1:1). Saturation is monitored by measuring the absorbance at 280 nm and the hemagglutinating activity of the solution before and after passage through the column. At saturation the column is washed extensively with PBS until the absorbance at 280 nm is below 0.050. Specific elution is effected by 0.005-0.015M D-GalNAc in PBS. The eluted material is concentrated by ultrafiltration and passed twice through a column of Biogel P-10 to remove free and bound n-GalNAc. Insoluble hog blood group A + H substance is prepared by copolymerization with the N-carboxyanhydride of L-leucinC as described by Kaplan and Kabat? The hemagglutinin binding capacity of polyleucyl hog blood group A + H substance is approximately 30% on a weight basis. 6 The crude extract contains in addition to Helix pomatia A hemagglutinin a second component6 (Fig. 1B), which also combines with hog blood group A + H substance. This protein, which does not agglutinate human A erythrocytes, can be removed by preeluting the column with 0.05 M D-Glc or by extensive washing with PBS. In a typical experiment~ 85.5% of the adsorbed hemagglutinin was recovered after elution with 0.005 M D-GalNAc. Very little additional nitrogen, however, was recovered by raising the D-GalNAc concentration to 0.05M (0.01%) or by elution with 2 M KSCN (0.7% of total eluted nitrogen). The latter fraction contained only nonhemagglutinin components, indicating that the hemagglutinin is completely eluted. The hemagglutinin can also be purified by adsorption to Sephadex G-200 and elution with D-GalNAc, D-Gal, or even D-Glc in the same manner as described for insoluble hog blood group A + H substance2 '1° H. Tsuyuki, H. von Kley, and M. A. Stahmann, J. Amer. Chem. Soc. 78, 764 (1956). s M. E. Kaplan and E. A. Kabat, J. Exp. Med. 123, 1061 (1966). O. Kiihnemund and W. KShler, Experientia 25, 1137 (1969). ,o I. Ishiyama and G. Uhlenbruck, Z. Klin. Chem. Klin. Biochem. 9, Heft 5 (1971).
370
PURIFICATION OF CARBOHYDRATE-BINDING PROTEINS
~.~
[45]
o.
6
z
4~
t28
~ u
E<
ooE ~
2
v
0
I'o
v
2' 0
v
' 30
4'0
' 5O
6 'o
~o
"-°
/.zg antigen added
Fla. 1. Ultraeentrifugal, immunoeleetrophoretic, and precipitin pattern of immunosorbent purified Helix pomatia A hemagglutinin. (A) Schlieren pattern of purified snail hemagglutinin [fraction II of S. HammarstrSm and E. A. Kabat, Biochemistry 8, 2696 (1969)] (1.30 mg of N per milliliter in 0.9% NaC1). The photomicrograph was taken after 144 minutes at 50740 rpm. (B) Immunoelectrophoretic analysis of purified snail hemagglutinin, fraction II, 1.30 mg of N per milliliter (center well), and of crude extract of albumin gland (upper and lower wells); rabbit antiserum against crude extract of albumin gland (upper trough) and hog blood group A + H substance (lower trough) were added to develop reactions. (C) Precipitation of p.urifled snail hemagglutinin fraction II, 6A7 gg of N per tube, by human blood group A substance (cyst MSM) ((~--O), The total volume was 200 #l. Hemagglutination titer of supernatants against human Al-erythrocytes (0--{}). I t is likely t h a t nonreducing a-linked D-Glc end groups in dextran are responsible for the binding of the hemagglutinin. This is a v e r y w e a k interaction since D-Glc can displace the hemagglutinin from the Sephadex column. Precipitin inhibition studies furthermore show t h a t D-Glc or Me-a-D-Glc are v e r y poor inhibitors as c o m p a r e d with D-GalNAc (see below). T h e a b u n d a n c e of D-Glc end groups in dextran and the high v a l e n c y of the hemagglutinin seem, however, to compensate for the w e a k -
[45]
SNAIL HEMAGGLUTININ
371
hess of the interaction. A similar situation has been described for certain group C antistreptoeoccal antibodies? ~ Purity Immunosorbent purified Helix pomatia A hemagglutinin is homogeneous on gel filtration on Biogel P-150 and P-300 and gives only one symmetrical peak in the analytical ultracentrifuge 6 (Fig. 1A). It gives only one line in immunoelectrophoresis even when tested at very high protein concentration with antiserum to the crude extract6 (Fig. 1B). With this antiserum at least 15 components were detected in the crude extract (Fig. 1B). It is furthermore completely precipitated by human blood group A substance 6 (Fig. 1C). Polyacrylamide gel electrophoresis at alkaline pH revealed, however, a certain degree of heterogeneity. Although approximately 90% of the material eluted with 0.005-0.015 M n-GalNAc appeared as one band, three additional bands with closely similar electrophoretic mobilities were seen. The relative amount of these bands was different in fractions eluted earlier from the immunosorbent column than in fractions eluted later. In the last fraction (1.3% of total eluted hemagglutinin) three bands (including the major band) of approximate equal density were obtained. Since this fraction, as well as the earlier fractions, were completely precipitated by human blood group A substance B it follows that all bands must represent the Helix pomatia A hemagglutinin. Polyacrylamide gel electrophoresis at different gel concentrations showed furthermore that these bands differed in charge but not in size2 This may either reflect a heterogeneity of the starting material--a pool of albumin glands from several hundred snails was used--or may be due to secondary chemical changes. Chemical and Physicochemical Properties The sO0,wvalue for the hemagglutinin was determined to 5.3 s (0.9% NaC1). From this, the intrinsic viscosity, and the partial specific volume, a molecular weight of 1.0 × 105 was calculated2 Further ultracentrifugation studies, 12 using the meniscus depletion sedimentation equilibrium method, showed that this value was too high. A mean molecular weight of 79,000 was obtained. TM Amino acid analysis shows that the hemagglutinin contains approximately 18 moles of half-cystine and 10 moles of methionine per molecu11T. J. Kindt, C. W. Todd, K. Eichmann and R. M. Krause, J. Exp. Med. 131, 343 (1970). ~S. HammarstrSm, A. West55, and I. BjSrk, Scand. J. Immunol. (1972) in press.
372
PURIFICATION OF CARBOHYDRATE-BINDING
PROTEINS
[45]
lar weight of 79,000. 6 Carbohydrate analysis revealed the presence of D-galactose (4.0%) and D-mannose (3.3%)2 Trace amounts of hexosamine were also detected. It is, however, likely that the latter represents residual D-GalNAc not completely removed by dialysis and gel filtration. Subunit Structure Direct alkylation of the hemagglutinin at pH 8.6 either in Tris buffer alone or in 6 M guanidine.HC1 showed that free sulfhydryl groups were absent 12 (Table I). Reduction with excess dithiothreitol (DTT) in 6 M guanidine.HC1 lead, on subsequent alkylation, to the incorporation of approximately 18 moles of [14C]-acetate per molecular weight of 79,00012 (Table I). Thus under these conditions the hemagglutinin was completely reduced. Gel filtration of the alkylated material on Sephadex G-100 in 6 M guanidine.HC1 gave the elution pattern shown in Fig. 2A. One symmetrical included peak, in addition to aggregated material in the exclusion volume, was obtained. 12 Under these conditions the unreduced hemagglutinin is included22 The mean molecular weight of the included component (Helix pomatia A hemagglutinin subunit) was 13,000 as determined from a calibration curve for the same column, using completely reduced and alkylated proteins of known molecular weights ~ (Fig. 2B). The amino acid composition showed that the hemagglutinin per molecular weight 13,000 contains 12 moles of lysine and arginine and 3 moles of half cystine. Thus, if the protein consists of identical subunits, 13 peptides with maximally 3 containing cysteine would be expected on tryptic digestion. In fact, 13-14 maior peptides (2 acid, 6 neutral, and 5 or 6 basic peptides) were found after combined electrophoretic and chromatographic analysis 13 of tryptic digest, prepared from reduced and ~4C-carboxymethylated or performic acid oxidized hemagglutinin. 12 Three of these peptides were radioactive. One of them (acid peptide) gave however a more intense radioactive spot. 12 These data suggest that the hemagglutinin consists of only one type of subunit with an approximate molecular weight of 13,000. Reduction in the absence of unfolding agents followed by alkylation revealed that disulfide bonds were not cleaved ~2 (Table I). Treatment of the hemagglutinin with 6 M guanidine.HCl at pH 4.0 or with 6 M guanidine.HC1 alone for 15 minutes up to 2 days gave one component with a molecular weight of 26,000-31,000 both on gel filtration in 6 M guanidine.HC1 + 0.1 M D-GNAc and on ultracentrifugation (meniscus depletion sedimentation equilibrium method).1_- Moreover the :a H. JSrnvall, Eur. J. Biochem. 16, 41 (1970).
[45]
SNAIL HEMAGGLUTININ
373
X
0
v
>
V
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~
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0
m
~
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0 Z 0
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I ~
.
374
PURIFICATION OF CARBOHYDRATE-BINDING PROTEINS
A
[45]
B
A280
0/-~0 Ribonuclease
0.30
..... ~ s o z y m e l
1600
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1.60
i
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=
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.
.
.
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|
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-~ConA
I
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i I
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10 20 30 40 _503 MOLECULARWEIGHT X 10
FIG. 2. Gel filtration profile of completely reduced and [14C]alkylated Helix pomatia A hemagglutinin on Sephadex G-100 column in 6 M guanidine'HC1 (A) and calibration curve for the same column (B). (A) The solid line denotes absorbance at 280 nm, and the dashed line denotes radioactivity. The column was 2.5 × 40 cm. The exclusion volume (Vo) is indicated in the figure. (B) Completely reduced and alkylated proteins were used for calibrations. The dashed lines denote the V~/Vo value and the molecular weight for the major component of Fig. 2A. Con A, concanavalin A.
hemagglutinin did not dissociate further when treated with 7 M guanidine.HC1 containing 1 M propionic acid as determined by gel filtration experiments in this medium.12 Control experiments demonstrated that the hemagglutinin was not reduced under these conditions (0.2 mole [14C]acetate per molecular weight 79,000). Conditions were also found that gave rise to partial reduction of the hemagglutinin (e.g., reduction with excess DTT in 8 M u r e a _ 0.1 M D-GNAc or in 6 M guanidine-HC1 containing 0.1-0.2M D-GalNAc or D-GNAc). 3-4 disulfide bonds were cleaved under these conditions12 (Table I). Gel filtration in dissociating agents in the presence of D-GNAc gave only one symmetrical component with a molecular weight of 12,50016,700 (determined from standard curves for reduced and unfolded proteins or non-reduced but unfolded proteins respectively). This component furthermore contained an active carbohydrate binding site. 12 Taken together these data suggest that the hemagglutinin is made up of six identical or closely similar polypeptide chains, each containing one intrachain disulfide bond and a carbohydrate binding site. The subunits are arranged in pairs in which they are linked together by a single disulfide bond. Native hemagglutinin is formed by the interaction of non-covalent forces between three dimers.
[45]
SNAIL HEMAGGLUTININ
375
Homogeneity of Carbohydrate BLnding Site A blood group A active pentasaccharide, ARL 0.52, a-D-GalNAc (i--> 3) - [q-L-Fuc- (i --> 2) ] -fl-D-Gal- (I ---> 4) -fl-D-GNAc- (I ~ 6) -3-hexenetetrol (s) labeled with tritium in the hexenetetrol residue I' was used in equilibrium dialysis experiments to investigate the binding properties of Helix pomatia A hemagglutinin. This structure constitutes the determinant that is responsible for precipitation of blood group A substance by the hemagglutinin (see below). Figure 3A demonstrates a linear relationship when the binding data 1~ are plotted according to Scatchard. 16 A heterogeneity index, a, of 1.04 was obtained when the binding data were plotted according to Sips distribution 17,18 (Fig. 3B). Thus, within the experimental error the binding sites are homogeneous. On the basis of a molecular weight of 1.0 × 105 for the hemagglutinin, the equilibrium dialysis data indicate that the hemagglutinin contains one combining site/molecular weight of 16,000-17,000.1~ This value is in reasonable agreement with the molecular weight of the subunit.
B
A
50 25 "T
%
2O
0
15 o~
-I
I0
o
I
60
I
L
L
t
2
3
tog c (pM)
Fro. 3. Equilibrium dialysis experiments with purified Helix pomatia A hemagglutinin and tritium-labeled A active pentasaccharide [3H]AR~ 0.52. The data are plotted both according to Scatehard (A) and according to Sips (B). Approximately 12 mg of snail protein was used for each determination. 1, C. Moreno and E. A. Kabat, J. Exp. Med. 129, 871 (1969). ~S. tIammarstrlim and E. A. Kabat, Biochemistry 10, 1684 (1971). 16G. Scatchard, Ann. N.Y. Acad. Sci. 51, 660 (1949). 1TR. Sips, J. Chem. Phys. 16, 490 (1948). ~SF. Karush, Advan. Immunol. 2, 1 (1962).
376
PURIFICATION OF CARBOHYDRATE-BINDING PROTEINS
[45]
Further evidence for homogeneity of binding sites has been obtained by equilibrium dialysis displacement experiments 15 (Fig. 4). In this experiment the capacities of the cross reactive haptens Me-a-D-GalNAc, Me-a-D-GNAc, and Et-fl-D-GNAc to displace the labeled A active pentasaccharide, [3H]ARL0.52, from the binding site were investigated. Linear displacement curves essentially parallel with the self-displacement curve, ARL0.52, were found in the plot. Thus over the range tested (from 10 to 70-80% displacement) there is no substantial heterogeneity of association constants for the binding of the displacing haptens. Specificity of Carbohydrate Binding Site The specificity of the binding site of Helix pomatia A hemagglutinin has been investigated by the following procedures. Equilibrium Dialysis and Displacement. The intrinsic association constant (Ko) for the interaction of the hemagglutinin site with Me-a-DGalNAc or the blood group A active pentasaccharide is 5.0 × 103 1/mole at 25 ° and pH 7.215 (Table II; Figs. 3 and 4). These compounds are the best inhibitors of precipitation studied so far. The Ko values for Me-a-D'5 (D 0 I,¢ O4
d
II0 O
I00
•
n
9O .J
E
80
,-~
70
Z
~ 6o u. 0 z o ~ er
~
50 40 30 2O
Z
uJ U Z 0 ¢J
IO I
-5 I0
I
I0
l
-4
I0
I
-3
I0
-2
FREE CONCENTRATION OF COMPETITOR(M)
FIG. 4. Equilibrium dialysis displacement experiments with unlabeled ligands. Approximately 12 mg of hemagglutinin and a concentration of ['H]ARL 0.52 which gives 110 × 1 0 - ' M bound radioactive h a p t e n were employed for each determination. Displacing haptens were ARL0.52 (O----O), self displacement curve; Me-a-D-GalNAc ( D - - D ) , M e - a - , - G N A c ( A - - A ) , and Et-fl-D-GNAe ( O - - O ) . The dashed horizontal line signifies the concentration of bound radioactive hapten in the absence of competitor.
[451
SNAIL HEMAGGLUTININ
377
GNAc and Et-fl-D-GNAc were approximately 4.5 and 27 times lower than for Me-a-D-GalNAc, respectively 1~ (Table I I ; Fig. 4). Although the intrinsic association constants for the best inhibitors are relatively low, they are of the same order of magnitude as for other carbohydrateanticarbohydrate systems. 1~,19,2° Inhibition of Precipitation. The most complete data on the specificity of the hemagglutinin site have been obtained by inhibition of precipitation using either human blood group A substance or Salmonella typhimurium SH 180 lipopolysaccharide (LPS) as test antigens2 ,1~,21 These two systems were used since they differ markedly in sensitivity. With the same amount of hemagglutinin, approximately 1000 nmoles of added D-GalNAc is needed for 50% inhibition in the former system whereas 25 nmoles of added v-GalNAc is required for the same degree of inhibition in the latter. The reason for this difference is that the hemagglutinin reacts with a-linked D-GalNAc in blood group A substance, ~ whereas the structure, a-D-Gal (1--)6)-D-Glc..., is "immunodominant" in S. typhimurium SH 180 LPS. 21 Table III shows the concentrations of various monosaccharides, methylglycosides, and oligosaccharides needed for 50% inhibition. The data are based on complete inhibition curves. In summary the results show: (1) Me-a-D-GalNAc is the best inhibitor of all compounds investigated so far. (2) In the series of a-DGalNAc containing oligosaccharides and derivatives, only the terminal nonreducing D-GalNAc residue seems to bind significantly to the site. This may indicate that the combining site is relatively small. (3) Me-a-DGalNAc is only about 4 times more active than Me-a-D-GNAc, indicatTABLE II INTRINSIC ASSOCIATION CONSTANTS (Ko) AND STANDARD FREE-ENERGY CHANGE (--AF °) FOR THE INTERACTION OF Helix pomatia A HEMAGGLUTININ WITH VARIOUS HAPTENS
AT p H
7.3 A N D 25.0 + 0.i °
Hapten
Ko X 10-a (M-1)
--AF ° (kcal/mole)
ARL 0.52a Me-a-D-GalNAc Me-a-D-GNAc Et-f~-D-GNAc
5.0 5.0 1.1 0.18
5.04 5.04 4.14 3.08
a See text. ~gL. L. So and I. J. Goldstein, J. Biol. Chem. 243, 2003 (1968). soM. Katz and A. M. Pappenheimer, Jr., J. Immunol. 103, 401 (1969). ~1S. HammarstrSm, A. A. Lindberg, and E. S. Robertson, Eur. J. Biochem. 255 274 (1972).
378
PURIFICATION OF CARBOHYDRATE-BINDING PROTEINS
[45]
T A B L E III CONCENTRATION OF MONOSACCHARIDES~ METHYLGLYCOSIDES~ AND OLIGOSACCHARIDES N E E D E D FOR 5 0 ~
I N H I B I T I O N OF P R E C I P I T A T I O N OF P U R I F I E D
Helix pomatia A
HEMAGGLUTININ WITH DIFFERENT CARBOHYDRATE ANTIGENSa Micromoles
for 50%
inhibition
of precipitation
of hemagglutinin With Inhibitor Me-a-D-GalNAc
blood
With
germfree
group A
rat colon
substance
antigen b
0.76
With
S. typhimurium SH
180 LPS
--
--
--
--
0.96
--
--
ART.0.52"
2.00
--
--
Ph-a-v-GalNAc
1.65o
--
--
o-{-p-NO2Ph-a-D-GalNAc
5.37g
--
a-D-GalNAc-(1
--* 3 ) - D - G a l
a-v-GalNAc,-(1
--~ 3 ) - ~ - v - G a l -
>0.14
(1 --* 3 ) - D - G N A c
Et-~-D-GalNAc ~ p-NO2Ph-B-D-GalNAc
~
> 3.60
--
--
> 0.99g
--
--
D-GaINAc d
1.65
D-GalNH2 •
> 9.73
v-GalNAc-oV
> 11.6
0.130
0.024
--
--
--
--
2--O-Ac-Me-a-D-Gal
--
0.050
--
2-O-Ac-Me-~-v-Gal
--
0.098
--
Me-a-D-GNAt
4.00
Et-~-D-GNAc D-GNAt
10.0 9.00
D-GNH~e
> 18.8
D-GNAc-ol /
> 11.6g
2-O-Ac-Me-a-v-GNAc 2-O-Ac-Me-~-D-GNAc Me-a-D-Gal a - D - G a l - ( 1 ---* 6 ) - D - G l c Me-~-D-Gal
--0.52 ---
0.070 -0.150 ---
--
1.60
--
--
3.50
--
> 10.9 -> 10.7
-> 5.40
7.00 3.20.
--
--
~ - D - G a l - ( 1 --, 3 ) - D - G a l N A c
>0.59
--
--
~ - D - G a l - ( 1 --~ 3 ) - D - G N A c
> 1.95
--
--
~ - D - G a I - ( 1 --~ 4 ) - D - G N A c
>2.09
--
~ - D - G a l - ( 1 --* 6 ) - D - G N A c D-Gal
> 1.31 >15.8
->2.40
--10.5
Me-~-D-Glc
>8.50
--
Me-~-D-Glc
> 17.9
--
24.1 --
n-Glc
> 18.4
--
40
Me-a-D-Man
> 39.0
--
55
D-Man
> 22.3
--
80 --
Me-a-D-ManNAc
> 5.32
--
Me-B-D-ManNAc
> 4.08
--
--
D-ManNAc
> 11.3
--
--
L-Fuc
> 15.8
--
> 150
[45]
SNAIL HEMAGGLUTININ
379
TABLE III (Continued) Micromoles for 50% inhibition of precipitation of hemagglutinin
Inhibitor #-L-Fuc-(1 --+ 3)-D-GNAc D-Xyl D-Ara S. typhimurium TV 160 core fragment S. typhimurium SH 180 core fragment
With blood With germfree With group A rat colon S. typhimurium substance antigenb SH 180 LPS > 1.79 -----
------
-95 > 150 0.70 4.50
Precipitation was performed in a total volume of 200 ~1 except for S. typhimurium SH 180 LPS where 400 ~1 was used. The following amounts of hemagglutinin and carbohydrate antigens (= equivalence point) were used: 12.8 I~g of human blood group A substance (cyst MSM) + 5.2 pg N of hemagglutinin; 11.8 I~g of germfree rat colon antigen -{- 3.5 ~g N of hemagglutinin; 88.7 ~g of S. typhimurium SH 180 LPS + 3.4 ~g N of hemagglutinin. b Rat colon antigen was obtained by phenol-water extraction of feces from germfree rats. The preparation was purified by ethanol precipitation (55-65% ethanol fraction) and treated with neuraminidase. c See text. d The capacities of these three compounds to give 50% inhibition of precipitation of human blood group H substance, first stage periodate oxidation and Smith degradation (cyst JS; 14.9 ~g) with the hemagglutinin (3.9 ~g N) were also studied [S. HammarstrSm and E. A. Kabat, Biochemistry 10, 1684 (1971)]. The concentrations needed were 0.031, 0.072, and 0.010 ~mole/200 ul for Et-f~-D-GalNAc, p-NO2Ph-f~-n-GalNAc, and D-GaINAc, respectively. D-GalNH2, 2-amino-2-deoxyoD-galactose; D-GNH2, 2-amino-2-deoxy-D-glucose. J D-GalNAc-ol, 2-acetamido-2-deoxy-])-galactitol, D-GNAc-ol, 2-acetamido-2-deoxyD-glucositol. a These values are calculated from inhibition data obtained with 9.4 j~g of human blood group A substance (cyst MSM) and 3.9 I~g N of hemagglutinin. With this amount of hemagglutinin 0.92 ~moles of D-GalNAc was needed for 50% inhibition as compared to 1.65 #moles in the system shown in the table. ing t h a t the steric o r i e n t a t i o n of the h y d r o x y l group on C - 4 is of lesser i m p o r t a n c e . (4) T h e presence of a n e q u a t o r i a l l y oriented N - a c e t y l or O - a c e t y l group on C - 2 is essential for strong b i n d i n g to t h e site. (5) All a - g l y c o s i d i c a l l y linked c o m p o u n d s b i n d more s t r o n g l y to the site t h a n the corresponding f l - l i n k e d derivatives. (6) Sugars t h a t a r e i n a n y w a y s t r u c t u r a l l y r e l a t e d to D-GalNAc can be shown to i n t e r a c t w i t h t h e h e m a g g l u t i n i n site p r o v i d e d the a s s a y s y s t e m is m a d e sufficiently sensitive. Direct Precipitation. H u m a n blood group A substance, desialized ovine s u b m a x i l l a r y m u e i n , group C streptococcal polysaccharide, hog blood
380
[45]
PURIFICATION OF CARBOHYDRATE-BINDING PROTEINS
group A + H substances, and purified intestinal mucin from germfree rat~ are precipitated by the hemagglutinin. 3,6,15 In these macromolecules, multiple nonreducing a-linked D-GalNAc end groups are exclusively or predominantly responsible for the interaction with the hemagglutinin site. For human blood group A substance this has been rigorously shown by means of an N-deacetylase from Clostridium tertium2 This enzyme specifically removes the N-acetyl group on nonreducing D-GalNAe of the blood group A determinant. 22 Enzyme treatment reduced the precipitating ability of blood group A substance to approximately one-tenth that of the original preparation e (Fig. 5A). Re-N-acetylation restored its precipitating ability completely e (Fig. 5A). The hemagglutinin also precipitates teichoic acids from Staphylococcus aureus containing a-linked D-GNAc nonreducing end groups, but not those with fl-linked nonreducing D-GNAc 6 (Fig. 5B). The content of a-D-GNAc containing teichoic acids could be determined quantitatively in strains that have a mixture of both by using the 3528 strain as reference (this strain contains teichoic acid with a-linked D-GNAc nonreducing end groups exclusively). The hemagglutinin did not precipitate group A streptococcal poly-
5,0
A
B
4,0 ~3.0 z~2~0 to x a
0
i
=
10
o
.
20
.
.
.
.
=
L
=
,
30 40 0 4 8 pg ANTIGEN ADDED
=
12
,
o
,
O
o- - ~ p ~ - -
16 20 24 28 4252
FIG. 5. Precipitation studies on hemagglutinin of fraction II of S. HammarstrSm and E. A. Kabat [Biochemistry 8, 2696 (1969)]. (A) Precipitation of purified Helix pomatla A hemagglutinin, 3~3 #g of N of fraction II, by human blood group A substance prior to (O) and after (O) treatment with blood group A N-deacetylating enzyme and of enzyme-treated and re-N-acetylated (A) A substance; total volume 200 ~l. (B) Precipitation of teichoic acids from different strains of Staphylococcus aureus (A, strain 3528; m, NYH-6; O, Copenhagen; ×, A1) and of S. albus (Prengel) (O) with purified snail hemagglutinin; 3.83 #g of N of fraction II, total volume of 200 #1. = D. M. Marcus, E. A. Kabat, and G. Schiffman, Biochemistry 3, 437 (1964).
[45]
SNAIL HEMAGGLUTININ
381
saccharide. 1~ In this polysaccharide D-GNAc is fl-linked to the rhamnosyl- (1--> 3)-rhamnose backbone. From these studies it would appear that macromolecules with sterically accessible nonreducing a-linked D-GNAc end groups, but not with fl-linked D-GNAc end groups, are precipitated by the hemagglutinin. The hemagglutinin also precipitates blood group substances of B, H, Le a, or precursor specificity,e The amount needed to precipitate a given amount of hemagglutinin varied within and between serotypes but was in all cases higher than that needed for blood group A substance. 6 Human blood group H substance also precipitates with the hemagglutinin after periodate oxidation and Smith degradation. The material was actually more active than untreated H-substanceY 3 A second cycle of oxidation and degradation (2nd periodate step) decreased the activity markedly. However, some activity was still left.23 The group(s) responsible for precipitation in blood group substances other than A is (are) not known. A possible candidate is a-D-GalNAc linked directly on serine or threonine in the peptide backboneY 4 In light of the data obtained with LPS from Salmonella typhimurium rough mutants (see below) other interpretations are however possible. Recent studies 21 have shown that lipopolysaccharides from certain rough mutants of S. typhimurium are precipitated by the hemagglutinin. While the LPS from the parent smooth strain was inactive, LPS from mutants of chemotype Ra and Rb and to some extent also chemotype SR were precipitated by the hemagglutinin. LPS from mutants of chemotypes Rc, Rd, Re as well as isolated lipid A were inactive. The reaction was immunologically specific and could also be demonstrated by independent techniques. Lipopolysaccharide core fragments prepared by weak acid hydrolysis and purified by gel filtration from the Rb mutant TV160 and the Ra mutant SH 180 both inhibited precipitation between the hemagglutinin and SH 180 LPS (Table III). The inhibition powers of these fragments were comparable to melibiose (a-D-Gal-(1 --->6)-D-Glc). The precipitating capacity of TV 160 lipopolysaccharide was abolished after treatment with the enzyme D-galactose oxidase. It was suggested that the structural element, a-D-Gal-(1 --> 6)-l)-Glc . . , present in all LPS from active mutants is the "immunodominant" structure responsible for precipitation. This finding is rather unexpected considering the poor inhibition obtained with Me-a-D-Gal or melibiose. However, in a situation where both reagents are polyvalent even very weak interactions may be sufficient for precipitation. ~' S. HammarstrSm and E. A. Kabat, unpublished observations. K. O. Lloyd and E. A. Kabat, Proc. Nat. Acad. Sci. U~g. 61, 1470 (1968).
382
PURIFICATION OF CARBOHYDRATE-BINDING PROTEINS
[45]
Utility Helix pomatia A hemagglutinin m a y be a useful tool for the detection of carbohydrate end groups on macromolecules and on cell surfaces. I t is already used routinely in blood group serology as a specific agglutinin for h u m a n A erythrocytesY 5 Its absolute specificity for h u m a n A erythrocytes as compared to B- and O-erythrocytes can be shown by tracelabeling it with 125iodine3 (Fig. 6). I t has furthermore been shown to be useful in group determination of fl-hemolytic streptococci. Thus group C and H streptococci are specifically agglutinated by the hemagglutinin. 2e Likewise Escherichia coli 026, 086, 0111, and 0126 are agglutinated b y the crude snail extract. 27 However, when used as a tool for the detection of particular carbohydrate end groups, mere demonstration of an interaction with an unknown macromolecule or cell is obviously not sufficient. C o m p l e m e n t a r y analysis is in most cases essential for identification of the end group. I n such analysis inhibition with haptens m a y be performed in order to compare the relative strengths of the reaction with an already established system involving the same anticarbohydrate reagent. The unknown macromolecule m a y furthermore be chemically or enzymatically treated to remove or modify the end group in question. The relatively broad
16
f
12 X
E OL
o-- AI
8
0
.~M
4 o
:
~.--,
o
2
e,
4 pg
o-GaI.NAc ,
6
~-B and 0
,.,
8
,
10
,-*,
,
12
[125I]HEEMAGGt.UTININ
Fie.. 6. Binding of radioactive Helix pomrltia A hemagglutinin to human A-, B-, and O-erythrocytes. Approximately 40 × 10~ cells were incubated with different amounts of 1-'SI-labeledhemagglutinin (specific activity = 2700 cpm/~g) in a volume of 2.0 ml. The cells were washed two times with PBS, and cell-bound radioactivity was determined by y-counting. 25C. HSgman, personal communications. '~ W. KShler and O. Prokop, Z. Immunol. Forsch. 133, 50 (1967). 27G. Uhlenbruck, 0. Prokop, and W. Haferland, Zentralbl. Bakteriol. I Orig. 199, 271 (1966).
[46]
EEL ANTI-H(O) GLOBULIN
383
specificity of the hemagglutinin tends to limit its usefulness in this respect. More useful may be the possibility to separate polysaccharides, glycoproteins, oligosaccharides or even cells with the aid of the hemagglutinin, made insoluble through polyeondensation or otherwise.
[46]
Eel S e r u m A n t i - H u m a n B l o o d - G r o u p H ( O ) P r o t e i n 1 B y PARIMAL R . DESAI a n d GEORG F. SPRINGER
Eel anti-human blood-group H(O) protein, a potent hemagglutinin of human blood-group 0 erythrocytes,2 is a 7 S globulin?,4 Originally it was thought that the combining sites of the eel antibody for bloodgroup H(O) specific structures are complementary to a-L-fucopyranose (6-deoxy-a-L-galactopyranose),~,6 but we found that monosaccharides with both L- and D-galactose configuration are complementary to these sites and function as inhibitory haptens provided they possess at least one methyl group on either C-3 or C-5. 7-1° Numerous other hexoses, pentoses and their derivatives are inactive. 7 The eel anti H(O) protein specifically precipitates not only with blood-group H(O) active macromolecules of human, animal, and plant origin, but surprisingly also with 3-O-methyl-D-fucose (D-digitalose) and 3-O-methyl-D-galactose.8,9,11 The minimum combining structure which shows inhibitory activity with the eel anti-H(O) antibody is smaller than a monosaccharide. It consists of a methyl substituent attached equatorially to a pyranose ring, an ether oxygen adjoining this methyl group, and an axial, oxygencarrying substituent cis to the methyl group on a contiguous C atom2 ,1° ' This investigation was supported by National Science Foundation Grant GB-8378. The Department is maintained by the Susan Rebecca Stone Fund for Immunochemistry Research. " S. Miyazaki, Nagasaki Idai Hoiqaku Gyoho 2, 542 (1930). A. Bezkorovainy, G. F. Springer, and P. R. Desai, Biochemistry 10, 3761 (1971). 4 G. F. Springer and P. R. Desai, Vox Sang. 18, 551 (1970). W. M. Watkins and W. T. J. Morgan, Nature (London) 169, 825 (1952). e R. Kuhn and H. G. Osman, Hoppe-Seyler's Z. Physiol. Chem. 303, 1 (1956). G. F. Springer and P. Williamson, Biochem. J. 85, 282 (1962). 8 G. F. Springer, P. R. Desai, and B. Kolecki, Biochemistry 3, 1076 (1964). 9 G. F. Springer, T. Takahashi, P. R. Desai, and B. J. Kolecki, Biochemistry 4, 2099
(1965). lop. R. Desai and G. F. Springer, Proc. lOth Congr. Int. Soc. Blood Trans]us., Stockholm, p. 500 (1965). 1~G. F. Springer and P. R. Desai, Biochemistry 10, 3749 (1971).