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Mechanism of agglutination of red blood cells by oyster hemolymph
JOURNAL
OF
INVEHTEBRATE
PATHOLOGY
Mechanism
of
Cells
523-530
9,
( 1967)
Agglutination
by
Oyster
JOSEPH E. MCDADE’) Department
of Biological Newark, Accepted
Red
Blood
Hemolymphl R. TRIPP
AND M.
Sciences, Delaware November
of
University 19711 14,
of Delaware,
1966
Hemolymph from normal oysters ( Crassostrea uirginica ) agglutinates the red blood cells of many vertebrate species. Inhibition tests indicate that the reactive site on human cells is the ABO blood group hapten; chemically different sites serve as points of attachment in other species. The reactive protein molecule requires calcium to agglutinate human cells but does not require calcium to agglutinate cells of nonhuman species. In the absence of calcium, hemagglutinin is heat-labile and is degraded to protein subunits with loss of biological activity. Presumably the intact complex molecule has the proper spatial orientation to attach to specific sites on 1-4 cells and to bind them in a lattice formation that results in agglutination.
INTRODUCTION
Substances that agglutinate cells of other species have been found in the body fluids of several invertebrates (Tyler, 1946), but little is known about the nature of these substances or the mechanisms by which they act. Several hemagglutinins in the hemolymph of mollusks have been reported. A specific agglutinin for rabbit red blood cells found in the serum of the snail Vizjiparus malleatus was shown to be protein (Cheng and Sanders, 1962), and Boyd et al. (1966) described a protein from Otalu (Helix) Zadea that specifically agglutinates human type A red cells. Cushing et al. (1963) reported a substance in the serum of Octopus bimaculatus that inhibited the action of human anti-A serum. ’ Supported by a grant from the U.S. Public Health Service (GM 12070). ’ Part of a dissertation submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree Doctor of Philosophy. 523
Johnson ( 1964) described a substance, probably a protein, in extracts of butter clams (Saxidomus giganteus) that specifically agglutinated human AI and AIB red blood cells. A protein hemagglutinin found in oyster (Crassostrea virginica) hemolymph has been reported recently (Tripp, 1966). This report describes additional properties of oyster hemagglutinin, characterizes the red blood cell combining sites, and proposes a possible mechanism for hemagglutination.
MATERIALS
AND METHODS
Collection of Hemolymph. Oysters were dredged from the Broadkill River, Lewes, Delaware, and stored dry at 4°C. The anterior half of the left valve was removed and hemolymph was taken from the heart, pericardium, and adductor muscle via a 22-gauge needle attached to a Pasteur pipette. The pooled hemolymph was centrifuged to remove debris, filtered through
524
MCDADE
a Millipore HA filter (0.45 cc), divided into aliquots of 10 ml or less, frozen, and stored at -20°C. Hemagghtinin Titration. Microtitrations were performed in V-shaped plastic wells (Cooke Engineering Co., Alexandria, Virginia) by serially diluting oyster hemolymph with buffered (pH 7.1) 2.3% sodium chloride. To 0.025 ml of diluted oyster hemolymph was added 0.025 ml of 2% red blood cells in 0.85% saline. In experiments designed to test the effect of ions on hemagglutination, selected ions (magnesium, calcium, and potassium) were added to the buffered saline diluent at a final concentration of 0.1%. All tests were incubated at room temperature (25°C) for 3 hr. Titers were recorded as the reciprocal of the last dilution of hemolymph that agglutinated red blood cells and so represent the agglutinating capacity of 0.025 ml of oyster hemolymph mixed with an equal volume of red cell suspension. Hemagglutination Inhibition. The following saccharides were tested for their ability to inhibit hemagglutination: galactosamine (General Biochemicals, Chagrin Falls, Ohio 44022 ) ; D ( - ) -arabinose, D ( - ) ribose, D( + ) -mannose, D( + ) -glucosamine, n-acetylglucosamine, n-acetyl-nL-fucose, galactosamine (Nutritional Biochemicals Corp., Cleveland, Ohio 44128); D(+)galactose, D(f)-xylose, L ( + ) -rhamnose (Sigma Chemical Co., St. Louis, Missouri 63118); and n-trehalose (Baltimore Biological Laboratories, Baltimore, Maryland), Five milligrams of saccharide in 0.050 ml of 0.85% saline were added to 0.025 ml of dilutions of hemolymph in microtiter wells. These mixtures were incubated at room temperature for % hr. Then 0.025 ml of a 4% suspension of red blood cells was added to the sugar-hemolymph mixtures, the mixtures incubated at room temperature for 3 hr, and the hemagglutinin titer recorded. Preparation of Antihemolymph Serum. Two rabbits were each injected with a 1 : 1 mixture of oyster hemolymph and Freund’s
AND
TBIPP
complete adjuvant (Difco Laboratories, Inc., Detroit, Michigan). Each rabbit was initially injected with a total of 2 ml of antigen (0.5 ml beneath the skin of the back at each of four different sites). Three weeks later, the rabbits were injected again with 2 ml of hemolymph-adjuvant mixture in the same manner. Two weeks following the second injection, the animals were bled and their combined sera frozen and stored at -20°C. The rabbit antihemolymph serum had a precipitin titer of 16 when tested with undiluted hemolymph in capillary tube precipitation tests. Rabbit anti-bovine-serum-albumin (antiBSA) serum was prepared by giving four subcutaneous injections of 0.5 ml of 5% BSA solution (Nutritional Biochemicals Corp. ) to three rabbits at 3-day intervals. Ten days after the last injection the rabbits were bIed, the sera frozen and stored at -20°C. Gel Diffusion. Ouchterlony doublediffusion tests were performed to compare the diffusion pattern of hemolymph with the pattern for bovine serum albumin. Ionagar No. 2 (Consolidated Laboratories, Inc., Chicago Heights, Illinois ) at a final concentration of 0.85% in 0.1 N sodium barbital buffer (pH 7.6) was used as a diffusion medium. Wells were placed 2 cm apart. The antigens (oyster hemolymph and BSA) were placed in separate wells. The anti-BSA and antihemolymph sera were mixed together in equal volumes and placed in the other reaction well. Both antigens were used at a final protein concentration of 2 mg/ml. Duplicate plates were prepared and incubated for 7 days at 4” or 25°C. Hemagglutinin Neutralization. Oyster hemolymph was mixed with an equal volume of antihemolymph serum and incubated overnight at 4°C. The precipitate was removed by centrifugation, and the hemolymph antiserum mixture tested for its ability to agglutinate sheep, rabbit, and human red blood cells. Hemolymph mixed
OYSTER
HEMAGGLUTININ
with normal rabbit serum was also tested for hemagglutinin activity and thus served as a control. Protein Determination. Protein determinations were by the Folin-Ciocalteau method of Oyama and Eagle ( 1956). Unknowns were compared to a standard curve that had been prepared with known concentrations of BSA. Dialysis. To determine the solubility characteristics of oyster hemagglutinin, 2to S-ml samples of hemolymph were dialvzed against 2 liters of cold ( 1°C ) distilled water for 3 days. To test the effect of various ions on hemagglutinin activity, hemolymph was dialyzed against cold 2.3% sodium chloride (pH 7.1) or against cold 2.3% sodium chloride supplemented with potassium chloride, magnesium chloride, or calcium chloride (all reagent grade) at a final concentration of 0.1%. Heat Treatment. One milliliter aliquots of hemolymph were placed in stoppered tubes and heated in a water bath at various temperatures (55-100°C) for 1 hr. The samples were cooled to room temperature and any coagulant removed by centrifugation. The heated samples were tested for hemagglutinin titers using the procedure outlined above. Ilydr0lysi.s Of Hemolymph. One milliliter of 0.1 N acetic acid was added to 1 ml of oyster hemolymph and the mixture heated at 60°C for 1 hr. The reactants were cooled to room temperature in an ice bath and then neutralized by the dropwise addition of 1 N sodium hydroxide. One drop of 0.02% phenol red was used as an indicator. Serially doubling dilutions (l/20 through l/640) of the hydrolyzed hemolymph were made with saline and these dilutions tested in gel diffusion tests with antihemolymph serum. RESULTS
A previous study (Tripp, 1966) demonstrated that oyster hemolymph would ag-
52.i
glutinate the red blood cells of several vertebrates (mouse, rabbit, guinea pig, chicken, sheep, horse, and ox). This study has extended this list to include a monkey ( Cercopitlzicus &hops), an amphibian ( Ranu pipiens ), and two fish, the winter flounder ( Pseudopleuronectes americanus 1, and the perch ( Roccus americanus). Agglutination has occurred with every types of red cell that has been tested and this suggested the existence of a receptor site common to all vertebrate red cells. In an attempt to identify this common receptor, hemagglutination inhibition tests were performed using 13 different sacchnrides (Table I), some of which arr kno\nsn to be associated with red blood cell antigens. It can be seen that no one sac&arid<% inhibited agglutination of every type of red cell. Agglutination of human red culls was completely inhibited by galactosamine, n-acetylglucosamine, and ,n-acetylgalactosamine and partially inhibited by glucosamine. Inhihition of agglutination of other types of erythrocytes by single sugars was not as pronounced, although agglutination of rabbit cells was completely inhibited bv ribose. Preliminary experiments demonstrated that human red blood cells were not agglutinated by oyster hemolymph with sodium citrate added. It was thought that this inhibitory effect might be related to the c-he-. lation of calcium ions. Therefore hemolymph was dialyzed against salt solutions of known ionic composition and tested for hemagglut inin activity ( Table 2 J . Shthep and rabbi{ red blood cells are agglutinated bv hemol>mph regardless of its ion contfmt, while human red blood cells are agglutinated only in the presence of calcium ion. Moreover, hemolymph that has lost the ability to agglutinate human red blood cells due to dialysis against sodium chloride regains its ability to agglutinate these cells if calcium ion iS: subsequently added. Calcium ion alone has no obvious ef&c.t on human erythrocytes. When hrmolymph
526
MC
DADE
AND
was dialyzed against distilled water, all hemagglutinating activity was lost, probably due to the precipitation of protein during dialysis. It is known that oyster hemagglutinin is inactivated at temperatures above 65°C (Tripp, 1966). In the present study, the effect of ions on the heat stability of the hemagglutinin was determined ( Fig. 1) . Oyster hemolymph dialyzed against calcium-containing salt solutions was inactivated at the same temperature as undialyzed hemolymph, while oyster hemolymph dialyzed against calcium-deficient
TRIPP
solutions was more heat-labile. Calcium is necessary for heat stability of the hemagglutinin molecule, and may be an integral part of that molecule. - Oyster hemolymph is readily precipitated by the addition of antihemolymph serum. The hemagglutinating ability of hemolymph is completely neutralized by the addition of antihemolymph serum (Table 3), while hemolymph mixed with normal rabbit serum retains its hemagglutinating capacity for sheep, rabbit, or human red blood cells. Figure 2 illustrates the precipitin pattern
TABLE FORMAT:
HEMAGGLUTINATION
1 INHIBITION
BY SACCHARIDES
Hemagglutinin Type Sugar
Human (A, 0, B)
added
None D-Galactosamine D( +)-Mannose D-( -)-Arabinose D( +)-Glucose D( +)-Galactose L( +)-Rhamnose D( +)-Glucosamine D( -)-Ribose D-Trehalose D(+)-XylOSe L-Fucose
n-Acetylglucosamine n-Acetyl-D-galactosamine
Horse
Mouse
32
16
32
0
16
8
256 64
32
32
16
32
256
64
64 64 64
32 32
16 16
64
16 16
256 256 64
64
32 33
32 32 32
64 64
64 16
32
64 32 64
64 64 64
64 64
64 64
64 64
64 64
64
64
4 32
8
32
256 256
16
32 32 32
I6 16 16
0 32
64 256
32 32
256 256
16 16
32 32
256 256
TABLE ON REn
CELL
Sheep Rabbit Human Human Human Human
of
A 0 B AB
IJndialyzed 12s 32 16 16 16 16
NaCl 128 32 0 0 0 0
NaCl
Sheep
64
2
AGGLUTINATION Hemolymph
RBC
cell:
Rabbit
0
OFIONS
of red blood
Monkey
0
EFFECT
titer
+ KC1
BY OYSTER
HEMOLYMPH
dialyzed NaCl
against
+ MgClz
123 32 0 0
128 32 0 0
0 0
0 0
NaCl
+ CaClz 123 32 16 16 16 16
OYSTER
obtained when BSA and oyster hemolymph react with their respective antibodies in agar gel. The oyster system had two bands, one approximately midway between the reactant wells, and another near 6
4
its antiserum. The major component is a slow moving fraction while a small amount of protein migrates at a slightly faster rate. Samples of hemolymph protein dialyzed against calcium-containing and calcium-deficient salt solutions [Figs. 3 ( b ) and 3(c), respectively] give patterns identical to those of the native material. However, if the dialyzed samples are heated at 60” for 1 hr differences in the precipitin patterns are seen. In the presence of calcium the normal pattern is maintained [Fig. 3( e ) ] but in the absence of calcium there is a marked decrease in the amount of the faster component [Fig. 3i cl )I. Fur-
.-. o--o
K 52 LLJ iG I-
8
t-
4 -c,- i
,\,
0 25
55
65
-o--k-o-.-&
75
527
HEMAGGLUTININ
100
85
TEMPERATURE FIG. stability
1. The effect of calcium of oyster hemagglutinin.
ion
on
the
heat
HEMOLYMPH
the antigen well. The BSA was precipitated near the antibody well, indicating that it has a lower molecular weight than the hemolymph proteins. Attempts to selectively remove one of the oyster precipitin bands by adsorbing hemolymph with sheep red blood cells were unsuccessful, although II% of the hemolymph protein was removed by this procedure. Hemolymph protein may be altered by the removal of calcium ions and heating. Figure 3(a) illustrates the pattern of precipitins formed when native hemolymph is diluted serially and allowed to react with
FIG. 2. Pattern of bovine serum albumin (BSA) and oyster hemolymph proteins precipitated by their respective antibodies in agar gel.
TABLE ~ITTRALIZATION
3
OF HEMAGGLUTINIX Oyster
- ..__
l’ype
RBC
Rabbit Sheep Human Human Human
A 0 B
Human
OR
Normal -
rabbit
'BY ANTISERCM hemolympb
plw
serum Antioyster _ .--~--~___-.
serum
3”
0
158 16
0
16 16
0 0
16
0
~.
0
I_---
528
MC
DADE
AND
ther, if the hemolymph is subjected to mild (0.1 N acetic acid) hydrolysis and then allowed to react with the antiserum [Fig. 3( f ) 1, the native protein nearly disappears. Apparently it is converted to the faster diffusing fraction. Stronger hydrolytic treatment completely abolishes the outer band. Attempts to purify oyster hemagglutinin by ammonium sulfate precipitation have been unsuccessful. Hemagglutinin is precipitable with ammonium sulfate, but it cannot be separated from other hemolymph proteins by this method. Similarly, electrophoresis of oyster hemolymph on polyacrylamide gel, starch gel, and cellulose acetate has failed to identify the hemagglutinin. DISCUSSION Oyster hemolymph agglutinates the red cells of all the species that have been tested. Since adsorption of hemolymph with
TRIPP
guinea pig antigen removes the agglutinin to some but not all types of red cells ( Tripp, 1966), the reactive site of the red cells is not Forssman antigen. That same study also showed that adsorption of hemolymph with boiled beef red cells removes the agglutinins to all types of erythrocytes, suggesting that oyster protein agglutinates various red cells by attaching to a common heat-stable receptor that is present in its entirety in beef cell stroma. To test this hypothesis, a series of sugars, some of which are known to be components of certain blood group haptens were tested for their ability to inhibit agglutination. The complete inhibition of human red cell agglutination by n-acetylglucosamine and n-acetyl-n-galactosamine suggests that agglutination of human red cells is by attachment to the ABO haptens, since these saccharides are part of a “backbone” molecule that is common to the ABO antigens (Watkins, 1966). Glucosamine and galactosamine also inhibit agglutination of hu-
@@@ (0,’
(b)
(c)
(d)
(e)
(f)
Fro. 3. The effect of various ions on the gel diffusion pattern of oyster hemolymph serum was placed in the center well, while dilutions of antigen wells. The protein concentrations (mg/ml) of antigen in each respective which represents the pattern of normal hemolymph; (b) and (c) show hemolymph dialyzed against calcium-containing and calcium-deficient salt and (e) indicate the pattern obtained when hemolymph dialyzed against calcium-containing (e) salt solutions are heated at 60°C for 1 hr; (f) shows hydrolyzed with 0.1 N acetic acid at 66°C for 1 hr.
hemolymph proteins. Antiwere placed in the outer well are indicated in (a), the pattern obtained with solutions, respectively; (d) calcium-deficient (d ) or the pattern of hemolymph
OYSTER
HEMAGGLUTININ
man cells and are structurally related to the acetyl-substituted sugars. The lack of complete inhibition of agglutination of cells of other species by these same saccharides shows that the reactive sites on red cells is not a single chemical entity. If agglutination of every type of red cell is caused by lattice formation with a single protein molecule, then that molecule must have the ability to combine with a whole variety of saccharides. It is possible that all of these saccharides might be part of a complex hapten determinant found in its entirety in beef red cells, but this seems unlikely when one considers the relative economy of the chemical structure of known antigens. A more likely possibility is that oyster hemolymph contains a family of protein molecules each with a slightly different specificity and capable of reacting with one or a few structurally similar sugars. The hemagglutin macromolecules presumably are stable aggregates of a number of these protein subunits and have the proper spatial arrangement to attach to different sites on red cells. This latter hypothesis is consistent with all data presently available on oyster hemagglutinin. It has been reported previously (Tripp, 1966) that oyster hemagglutinin is a protein and further characterization of this protein is now possible. It is precipitable with ammonium sulfate but apparently the agglutinin is not sufliciently different from other hemolymph proteins to be separable by this method. It is insoluble in distilled water and therefore may be an euglobulin. It is antigenic since antibodies formed by rabbits precipitate the antigen and neutralize its agglutinative properties. Precipitation of hemolymph with its specific antibody shows that its rate of diffusion in agar is much slower than bovine serum albumin. Oyster hemolymph protein must therefore be of considerably greater molecular weight than 69,000, the value attributed to bovine serum albumin. Electrophoretic
529
mobility data (Woods et al., 1958; Gaumer, personal communication) show that oyster hemolymph protein is a large molecule of small net charge. Attempts have been made to associate hemagglutinin with a specific precipitin band by adsorbing hemolymph with sheep cells before testing it with antiserum in gel diffusion tests. These attempts have failed because only about 11% of the hemolymph protein is removed by the most intensive adsorption procedure and the precipitin system that was used cannot detect ir change of this magnitude. However, the: removal of protein by this adsorption dots support the hypothesis that hemagglutinin is a protein. The double-diffusion precipitin tests rc’ported here suggest strongly that the hcmagglutinin is associated with the slowlv diffusing protein component of oyster hemolymph. When calcium is removed a sulrtle change in structure results in the inability of the protein to bind human cells, but nonhuman cells can still be bound in lattice formations and agglutination results. The calcium-deficient molecule is more sensitive to heat and apparently is &Igraded to protein subunits with ‘concomitant loss of biological activity. This cvidence also suggests that normal oyster hemolymph contains a family of proteins associated as a stable macromolecule (hemagglutinin) . More detailed studies of the physical and chemical properties of oyster hemolymph protein. The significance of this hemagglutinin is obscure but several possible explanations may be considered: ( 1) Boyd ( 1962) has proposed that hemagglutinins that occur naturally in plants (lectins) function in the mobilization of saccharides for carbohydrate metabolism. Such a role in the oyster is possible since this animal is an herbivore that must digest, transport, and store various sugar molecules. (2) The apparentl) critical role of calcium in maintaining the
530
MCDADE
integrity of the hemagglutinin molecule suggests that it may b&row, or at some time in the evolutionary past, may have been, used for transportation of calcium ions for shell deposition. De Waele (cited in Galtsoff, 1964) postulated that such a protein-calcium carbonate complex was responsible for moblization of calcium ion for shell repair. (3) The ability of hemolymph to opsonize foreign material (Tripp, 1966) indicates that it may serve as part of a defense mechanism. The hemagglutinin could be a primitive type of “antibody molecule” capable of combining with many types of naturally occurring antigens. It is known, for example, that sugars that make up some bacterial antigens (Boyd, 1962; Salton, 1964) combine with oyster hemagglutinin. If this material should combine with the intact bacteria it might serve as a “recognition factor”-a type of natural antibody-that would enable the organism to distinguish between “self” and “not self” materials in the tissues and to remove foreign substances (Boyden, 1966). Such factors could confer a selective advantage on those animals that produce it. Further studies of the precise nature and origin of oyster hemagglutinin would, of course, permit a more definite statement of the importance of this substance. v
,
L
REFEI~EN~~~ BOYD,
“Introduction W. C. 1962. chemica1 Specificity,” 158 New York.
pp.
to ImmunoInterscience,
AND
TRIPP
BOYD,
W. C., BROWN, REBECCA, AND BOYD, L. G. 1966. Agglutinins for human erythrocytes in mollusks. J. Immunol., 96, 301-303. BOYDEN, S. V. 1966. Natural antibodies and the immune response. Aduan. Immzmol., 5, l-28. CHENG, T. C., AND SANDERS, B. G. 1962. Internal defense mechanism in molluscs and an electrophoretic analysis of a naturally occurring serum hemagglutinin in Viuiparus malleatus Reeve. Proc. Penna. Acad. Sci., 36, 72-83. GUSHING, J. E., CALAPRICE, NORA L., AND TRUMP, G. 1963. Blood group reactive substances in some marine invertebrates. Biol. Bull., 125, 69-80. GALTSOFF, P. S. 1964. The American Oyster Crassostrea virginica ( Gmelin). U.S. Fish Wildlife Serv., Fishery Bull., p. 95. JOHNSON, H. 1964. Human blood A1 specific agglutinin of the butter clam Saxidomus giganteus. Science, 146, 548-549. OYAMA, V. I., AND EAGLE, H. 1956. Measurement of cell growth in tissue culture with a phenol reagent ( Folin-Ciocalteau). PTOC. Sot. Exptk, BioE. Med., 91, 305307. SALTON, M. R. J. 1964. “The Bacterial Cell Wall,” 293 pp. Elsevier Co., New York. TRIPP, M. R. 1966. Hemagglutinin in the blood of the oyster Crassostrea uirginica. J. Invertebrate Pathol., 8, 478484. TRIPP, M. R., BISIGNANI, L. A., AND KENNY, M. T. 1966. Oyster amoebocytes in vitro. J. Znvertebrate Pathol., 8, 137-140. TYLER, A. 1946. Natural heteroagglutinins in the body fluids and seminal fluids of various invertebrates. Biol. Bull., 90, 213-219. WATKINS, WINIFRED M. 1966. Blood group substances. Science, 152, 172-181. WOODS, K. R., PAULSEN, E. C., ENGLE, R. L., AND PERT, J. H. 1958. Starch gel electrophoresis of some invertebrate sera. Science, 127, 519-520.
OF
INVEHTEBRATE
PATHOLOGY
Mechanism
of
Cells
523-530
9,
( 1967)
Agglutination
by
Oyster
JOSEPH E. MCDADE’) Department
of Biological Newark, Accepted
Red
Blood
Hemolymphl R. TRIPP
AND M.
Sciences, Delaware November
of
University 19711 14,
of Delaware,
1966
Hemolymph from normal oysters ( Crassostrea uirginica ) agglutinates the red blood cells of many vertebrate species. Inhibition tests indicate that the reactive site on human cells is the ABO blood group hapten; chemically different sites serve as points of attachment in other species. The reactive protein molecule requires calcium to agglutinate human cells but does not require calcium to agglutinate cells of nonhuman species. In the absence of calcium, hemagglutinin is heat-labile and is degraded to protein subunits with loss of biological activity. Presumably the intact complex molecule has the proper spatial orientation to attach to specific sites on 1-4 cells and to bind them in a lattice formation that results in agglutination.
INTRODUCTION
Substances that agglutinate cells of other species have been found in the body fluids of several invertebrates (Tyler, 1946), but little is known about the nature of these substances or the mechanisms by which they act. Several hemagglutinins in the hemolymph of mollusks have been reported. A specific agglutinin for rabbit red blood cells found in the serum of the snail Vizjiparus malleatus was shown to be protein (Cheng and Sanders, 1962), and Boyd et al. (1966) described a protein from Otalu (Helix) Zadea that specifically agglutinates human type A red cells. Cushing et al. (1963) reported a substance in the serum of Octopus bimaculatus that inhibited the action of human anti-A serum. ’ Supported by a grant from the U.S. Public Health Service (GM 12070). ’ Part of a dissertation submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree Doctor of Philosophy. 523
Johnson ( 1964) described a substance, probably a protein, in extracts of butter clams (Saxidomus giganteus) that specifically agglutinated human AI and AIB red blood cells. A protein hemagglutinin found in oyster (Crassostrea virginica) hemolymph has been reported recently (Tripp, 1966). This report describes additional properties of oyster hemagglutinin, characterizes the red blood cell combining sites, and proposes a possible mechanism for hemagglutination.
MATERIALS
AND METHODS
Collection of Hemolymph. Oysters were dredged from the Broadkill River, Lewes, Delaware, and stored dry at 4°C. The anterior half of the left valve was removed and hemolymph was taken from the heart, pericardium, and adductor muscle via a 22-gauge needle attached to a Pasteur pipette. The pooled hemolymph was centrifuged to remove debris, filtered through
524
MCDADE
a Millipore HA filter (0.45 cc), divided into aliquots of 10 ml or less, frozen, and stored at -20°C. Hemagghtinin Titration. Microtitrations were performed in V-shaped plastic wells (Cooke Engineering Co., Alexandria, Virginia) by serially diluting oyster hemolymph with buffered (pH 7.1) 2.3% sodium chloride. To 0.025 ml of diluted oyster hemolymph was added 0.025 ml of 2% red blood cells in 0.85% saline. In experiments designed to test the effect of ions on hemagglutination, selected ions (magnesium, calcium, and potassium) were added to the buffered saline diluent at a final concentration of 0.1%. All tests were incubated at room temperature (25°C) for 3 hr. Titers were recorded as the reciprocal of the last dilution of hemolymph that agglutinated red blood cells and so represent the agglutinating capacity of 0.025 ml of oyster hemolymph mixed with an equal volume of red cell suspension. Hemagglutination Inhibition. The following saccharides were tested for their ability to inhibit hemagglutination: galactosamine (General Biochemicals, Chagrin Falls, Ohio 44022 ) ; D ( - ) -arabinose, D ( - ) ribose, D( + ) -mannose, D( + ) -glucosamine, n-acetylglucosamine, n-acetyl-nL-fucose, galactosamine (Nutritional Biochemicals Corp., Cleveland, Ohio 44128); D(+)galactose, D(f)-xylose, L ( + ) -rhamnose (Sigma Chemical Co., St. Louis, Missouri 63118); and n-trehalose (Baltimore Biological Laboratories, Baltimore, Maryland), Five milligrams of saccharide in 0.050 ml of 0.85% saline were added to 0.025 ml of dilutions of hemolymph in microtiter wells. These mixtures were incubated at room temperature for % hr. Then 0.025 ml of a 4% suspension of red blood cells was added to the sugar-hemolymph mixtures, the mixtures incubated at room temperature for 3 hr, and the hemagglutinin titer recorded. Preparation of Antihemolymph Serum. Two rabbits were each injected with a 1 : 1 mixture of oyster hemolymph and Freund’s
AND
TBIPP
complete adjuvant (Difco Laboratories, Inc., Detroit, Michigan). Each rabbit was initially injected with a total of 2 ml of antigen (0.5 ml beneath the skin of the back at each of four different sites). Three weeks later, the rabbits were injected again with 2 ml of hemolymph-adjuvant mixture in the same manner. Two weeks following the second injection, the animals were bled and their combined sera frozen and stored at -20°C. The rabbit antihemolymph serum had a precipitin titer of 16 when tested with undiluted hemolymph in capillary tube precipitation tests. Rabbit anti-bovine-serum-albumin (antiBSA) serum was prepared by giving four subcutaneous injections of 0.5 ml of 5% BSA solution (Nutritional Biochemicals Corp. ) to three rabbits at 3-day intervals. Ten days after the last injection the rabbits were bIed, the sera frozen and stored at -20°C. Gel Diffusion. Ouchterlony doublediffusion tests were performed to compare the diffusion pattern of hemolymph with the pattern for bovine serum albumin. Ionagar No. 2 (Consolidated Laboratories, Inc., Chicago Heights, Illinois ) at a final concentration of 0.85% in 0.1 N sodium barbital buffer (pH 7.6) was used as a diffusion medium. Wells were placed 2 cm apart. The antigens (oyster hemolymph and BSA) were placed in separate wells. The anti-BSA and antihemolymph sera were mixed together in equal volumes and placed in the other reaction well. Both antigens were used at a final protein concentration of 2 mg/ml. Duplicate plates were prepared and incubated for 7 days at 4” or 25°C. Hemagglutinin Neutralization. Oyster hemolymph was mixed with an equal volume of antihemolymph serum and incubated overnight at 4°C. The precipitate was removed by centrifugation, and the hemolymph antiserum mixture tested for its ability to agglutinate sheep, rabbit, and human red blood cells. Hemolymph mixed
OYSTER
HEMAGGLUTININ
with normal rabbit serum was also tested for hemagglutinin activity and thus served as a control. Protein Determination. Protein determinations were by the Folin-Ciocalteau method of Oyama and Eagle ( 1956). Unknowns were compared to a standard curve that had been prepared with known concentrations of BSA. Dialysis. To determine the solubility characteristics of oyster hemagglutinin, 2to S-ml samples of hemolymph were dialvzed against 2 liters of cold ( 1°C ) distilled water for 3 days. To test the effect of various ions on hemagglutinin activity, hemolymph was dialyzed against cold 2.3% sodium chloride (pH 7.1) or against cold 2.3% sodium chloride supplemented with potassium chloride, magnesium chloride, or calcium chloride (all reagent grade) at a final concentration of 0.1%. Heat Treatment. One milliliter aliquots of hemolymph were placed in stoppered tubes and heated in a water bath at various temperatures (55-100°C) for 1 hr. The samples were cooled to room temperature and any coagulant removed by centrifugation. The heated samples were tested for hemagglutinin titers using the procedure outlined above. Ilydr0lysi.s Of Hemolymph. One milliliter of 0.1 N acetic acid was added to 1 ml of oyster hemolymph and the mixture heated at 60°C for 1 hr. The reactants were cooled to room temperature in an ice bath and then neutralized by the dropwise addition of 1 N sodium hydroxide. One drop of 0.02% phenol red was used as an indicator. Serially doubling dilutions (l/20 through l/640) of the hydrolyzed hemolymph were made with saline and these dilutions tested in gel diffusion tests with antihemolymph serum. RESULTS
A previous study (Tripp, 1966) demonstrated that oyster hemolymph would ag-
52.i
glutinate the red blood cells of several vertebrates (mouse, rabbit, guinea pig, chicken, sheep, horse, and ox). This study has extended this list to include a monkey ( Cercopitlzicus &hops), an amphibian ( Ranu pipiens ), and two fish, the winter flounder ( Pseudopleuronectes americanus 1, and the perch ( Roccus americanus). Agglutination has occurred with every types of red cell that has been tested and this suggested the existence of a receptor site common to all vertebrate red cells. In an attempt to identify this common receptor, hemagglutination inhibition tests were performed using 13 different sacchnrides (Table I), some of which arr kno\nsn to be associated with red blood cell antigens. It can be seen that no one sac&arid<% inhibited agglutination of every type of red cell. Agglutination of human red culls was completely inhibited by galactosamine, n-acetylglucosamine, and ,n-acetylgalactosamine and partially inhibited by glucosamine. Inhihition of agglutination of other types of erythrocytes by single sugars was not as pronounced, although agglutination of rabbit cells was completely inhibited bv ribose. Preliminary experiments demonstrated that human red blood cells were not agglutinated by oyster hemolymph with sodium citrate added. It was thought that this inhibitory effect might be related to the c-he-. lation of calcium ions. Therefore hemolymph was dialyzed against salt solutions of known ionic composition and tested for hemagglut inin activity ( Table 2 J . Shthep and rabbi{ red blood cells are agglutinated bv hemol>mph regardless of its ion contfmt, while human red blood cells are agglutinated only in the presence of calcium ion. Moreover, hemolymph that has lost the ability to agglutinate human red blood cells due to dialysis against sodium chloride regains its ability to agglutinate these cells if calcium ion iS: subsequently added. Calcium ion alone has no obvious ef&c.t on human erythrocytes. When hrmolymph
526
MC
DADE
AND
was dialyzed against distilled water, all hemagglutinating activity was lost, probably due to the precipitation of protein during dialysis. It is known that oyster hemagglutinin is inactivated at temperatures above 65°C (Tripp, 1966). In the present study, the effect of ions on the heat stability of the hemagglutinin was determined ( Fig. 1) . Oyster hemolymph dialyzed against calcium-containing salt solutions was inactivated at the same temperature as undialyzed hemolymph, while oyster hemolymph dialyzed against calcium-deficient
TRIPP
solutions was more heat-labile. Calcium is necessary for heat stability of the hemagglutinin molecule, and may be an integral part of that molecule. - Oyster hemolymph is readily precipitated by the addition of antihemolymph serum. The hemagglutinating ability of hemolymph is completely neutralized by the addition of antihemolymph serum (Table 3), while hemolymph mixed with normal rabbit serum retains its hemagglutinating capacity for sheep, rabbit, or human red blood cells. Figure 2 illustrates the precipitin pattern
TABLE FORMAT:
HEMAGGLUTINATION
1 INHIBITION
BY SACCHARIDES
Hemagglutinin Type Sugar
Human (A, 0, B)
added
None D-Galactosamine D( +)-Mannose D-( -)-Arabinose D( +)-Glucose D( +)-Galactose L( +)-Rhamnose D( +)-Glucosamine D( -)-Ribose D-Trehalose D(+)-XylOSe L-Fucose
n-Acetylglucosamine n-Acetyl-D-galactosamine
Horse
Mouse
32
16
32
0
16
8
256 64
32
32
16
32
256
64
64 64 64
32 32
16 16
64
16 16
256 256 64
64
32 33
32 32 32
64 64
64 16
32
64 32 64
64 64 64
64 64
64 64
64 64
64 64
64
64
4 32
8
32
256 256
16
32 32 32
I6 16 16
0 32
64 256
32 32
256 256
16 16
32 32
256 256
TABLE ON REn
CELL
Sheep Rabbit Human Human Human Human
of
A 0 B AB
IJndialyzed 12s 32 16 16 16 16
NaCl 128 32 0 0 0 0
NaCl
Sheep
64
2
AGGLUTINATION Hemolymph
RBC
cell:
Rabbit
0
OFIONS
of red blood
Monkey
0
EFFECT
titer
+ KC1
BY OYSTER
HEMOLYMPH
dialyzed NaCl
against
+ MgClz
123 32 0 0
128 32 0 0
0 0
0 0
NaCl
+ CaClz 123 32 16 16 16 16
OYSTER
obtained when BSA and oyster hemolymph react with their respective antibodies in agar gel. The oyster system had two bands, one approximately midway between the reactant wells, and another near 6
4
its antiserum. The major component is a slow moving fraction while a small amount of protein migrates at a slightly faster rate. Samples of hemolymph protein dialyzed against calcium-containing and calcium-deficient salt solutions [Figs. 3 ( b ) and 3(c), respectively] give patterns identical to those of the native material. However, if the dialyzed samples are heated at 60” for 1 hr differences in the precipitin patterns are seen. In the presence of calcium the normal pattern is maintained [Fig. 3( e ) ] but in the absence of calcium there is a marked decrease in the amount of the faster component [Fig. 3i cl )I. Fur-
.-. o--o
K 52 LLJ iG I-
8
t-
4 -c,- i
,\,
0 25
55
65
-o--k-o-.-&
75
527
HEMAGGLUTININ
100
85
TEMPERATURE FIG. stability
1. The effect of calcium of oyster hemagglutinin.
ion
on
the
heat
HEMOLYMPH
the antigen well. The BSA was precipitated near the antibody well, indicating that it has a lower molecular weight than the hemolymph proteins. Attempts to selectively remove one of the oyster precipitin bands by adsorbing hemolymph with sheep red blood cells were unsuccessful, although II% of the hemolymph protein was removed by this procedure. Hemolymph protein may be altered by the removal of calcium ions and heating. Figure 3(a) illustrates the pattern of precipitins formed when native hemolymph is diluted serially and allowed to react with
FIG. 2. Pattern of bovine serum albumin (BSA) and oyster hemolymph proteins precipitated by their respective antibodies in agar gel.
TABLE ~ITTRALIZATION
3
OF HEMAGGLUTINIX Oyster
- ..__
l’ype
RBC
Rabbit Sheep Human Human Human
A 0 B
Human
OR
Normal -
rabbit
'BY ANTISERCM hemolympb
plw
serum Antioyster _ .--~--~___-.
serum
3”
0
158 16
0
16 16
0 0
16
0
~.
0
I_---
528
MC
DADE
AND
ther, if the hemolymph is subjected to mild (0.1 N acetic acid) hydrolysis and then allowed to react with the antiserum [Fig. 3( f ) 1, the native protein nearly disappears. Apparently it is converted to the faster diffusing fraction. Stronger hydrolytic treatment completely abolishes the outer band. Attempts to purify oyster hemagglutinin by ammonium sulfate precipitation have been unsuccessful. Hemagglutinin is precipitable with ammonium sulfate, but it cannot be separated from other hemolymph proteins by this method. Similarly, electrophoresis of oyster hemolymph on polyacrylamide gel, starch gel, and cellulose acetate has failed to identify the hemagglutinin. DISCUSSION Oyster hemolymph agglutinates the red cells of all the species that have been tested. Since adsorption of hemolymph with
TRIPP
guinea pig antigen removes the agglutinin to some but not all types of red cells ( Tripp, 1966), the reactive site of the red cells is not Forssman antigen. That same study also showed that adsorption of hemolymph with boiled beef red cells removes the agglutinins to all types of erythrocytes, suggesting that oyster protein agglutinates various red cells by attaching to a common heat-stable receptor that is present in its entirety in beef cell stroma. To test this hypothesis, a series of sugars, some of which are known to be components of certain blood group haptens were tested for their ability to inhibit agglutination. The complete inhibition of human red cell agglutination by n-acetylglucosamine and n-acetyl-n-galactosamine suggests that agglutination of human red cells is by attachment to the ABO haptens, since these saccharides are part of a “backbone” molecule that is common to the ABO antigens (Watkins, 1966). Glucosamine and galactosamine also inhibit agglutination of hu-
@@@ (0,’
(b)
(c)
(d)
(e)
(f)
Fro. 3. The effect of various ions on the gel diffusion pattern of oyster hemolymph serum was placed in the center well, while dilutions of antigen wells. The protein concentrations (mg/ml) of antigen in each respective which represents the pattern of normal hemolymph; (b) and (c) show hemolymph dialyzed against calcium-containing and calcium-deficient salt and (e) indicate the pattern obtained when hemolymph dialyzed against calcium-containing (e) salt solutions are heated at 60°C for 1 hr; (f) shows hydrolyzed with 0.1 N acetic acid at 66°C for 1 hr.
hemolymph proteins. Antiwere placed in the outer well are indicated in (a), the pattern obtained with solutions, respectively; (d) calcium-deficient (d ) or the pattern of hemolymph
OYSTER
HEMAGGLUTININ
man cells and are structurally related to the acetyl-substituted sugars. The lack of complete inhibition of agglutination of cells of other species by these same saccharides shows that the reactive sites on red cells is not a single chemical entity. If agglutination of every type of red cell is caused by lattice formation with a single protein molecule, then that molecule must have the ability to combine with a whole variety of saccharides. It is possible that all of these saccharides might be part of a complex hapten determinant found in its entirety in beef red cells, but this seems unlikely when one considers the relative economy of the chemical structure of known antigens. A more likely possibility is that oyster hemolymph contains a family of protein molecules each with a slightly different specificity and capable of reacting with one or a few structurally similar sugars. The hemagglutin macromolecules presumably are stable aggregates of a number of these protein subunits and have the proper spatial arrangement to attach to different sites on red cells. This latter hypothesis is consistent with all data presently available on oyster hemagglutinin. It has been reported previously (Tripp, 1966) that oyster hemagglutinin is a protein and further characterization of this protein is now possible. It is precipitable with ammonium sulfate but apparently the agglutinin is not sufliciently different from other hemolymph proteins to be separable by this method. It is insoluble in distilled water and therefore may be an euglobulin. It is antigenic since antibodies formed by rabbits precipitate the antigen and neutralize its agglutinative properties. Precipitation of hemolymph with its specific antibody shows that its rate of diffusion in agar is much slower than bovine serum albumin. Oyster hemolymph protein must therefore be of considerably greater molecular weight than 69,000, the value attributed to bovine serum albumin. Electrophoretic
529
mobility data (Woods et al., 1958; Gaumer, personal communication) show that oyster hemolymph protein is a large molecule of small net charge. Attempts have been made to associate hemagglutinin with a specific precipitin band by adsorbing hemolymph with sheep cells before testing it with antiserum in gel diffusion tests. These attempts have failed because only about 11% of the hemolymph protein is removed by the most intensive adsorption procedure and the precipitin system that was used cannot detect ir change of this magnitude. However, the: removal of protein by this adsorption dots support the hypothesis that hemagglutinin is a protein. The double-diffusion precipitin tests rc’ported here suggest strongly that the hcmagglutinin is associated with the slowlv diffusing protein component of oyster hemolymph. When calcium is removed a sulrtle change in structure results in the inability of the protein to bind human cells, but nonhuman cells can still be bound in lattice formations and agglutination results. The calcium-deficient molecule is more sensitive to heat and apparently is &Igraded to protein subunits with ‘concomitant loss of biological activity. This cvidence also suggests that normal oyster hemolymph contains a family of proteins associated as a stable macromolecule (hemagglutinin) . More detailed studies of the physical and chemical properties of oyster hemolymph protein. The significance of this hemagglutinin is obscure but several possible explanations may be considered: ( 1) Boyd ( 1962) has proposed that hemagglutinins that occur naturally in plants (lectins) function in the mobilization of saccharides for carbohydrate metabolism. Such a role in the oyster is possible since this animal is an herbivore that must digest, transport, and store various sugar molecules. (2) The apparentl) critical role of calcium in maintaining the
530
MCDADE
integrity of the hemagglutinin molecule suggests that it may b&row, or at some time in the evolutionary past, may have been, used for transportation of calcium ions for shell deposition. De Waele (cited in Galtsoff, 1964) postulated that such a protein-calcium carbonate complex was responsible for moblization of calcium ion for shell repair. (3) The ability of hemolymph to opsonize foreign material (Tripp, 1966) indicates that it may serve as part of a defense mechanism. The hemagglutinin could be a primitive type of “antibody molecule” capable of combining with many types of naturally occurring antigens. It is known, for example, that sugars that make up some bacterial antigens (Boyd, 1962; Salton, 1964) combine with oyster hemagglutinin. If this material should combine with the intact bacteria it might serve as a “recognition factor”-a type of natural antibody-that would enable the organism to distinguish between “self” and “not self” materials in the tissues and to remove foreign substances (Boyden, 1966). Such factors could confer a selective advantage on those animals that produce it. Further studies of the precise nature and origin of oyster hemagglutinin would, of course, permit a more definite statement of the importance of this substance. v
,
L
REFEI~EN~~~ BOYD,
“Introduction W. C. 1962. chemica1 Specificity,” 158 New York.
pp.
to ImmunoInterscience,
AND
TRIPP
BOYD,
W. C., BROWN, REBECCA, AND BOYD, L. G. 1966. Agglutinins for human erythrocytes in mollusks. J. Immunol., 96, 301-303. BOYDEN, S. V. 1966. Natural antibodies and the immune response. Aduan. Immzmol., 5, l-28. CHENG, T. C., AND SANDERS, B. G. 1962. Internal defense mechanism in molluscs and an electrophoretic analysis of a naturally occurring serum hemagglutinin in Viuiparus malleatus Reeve. Proc. Penna. Acad. Sci., 36, 72-83. GUSHING, J. E., CALAPRICE, NORA L., AND TRUMP, G. 1963. Blood group reactive substances in some marine invertebrates. Biol. Bull., 125, 69-80. GALTSOFF, P. S. 1964. The American Oyster Crassostrea virginica ( Gmelin). U.S. Fish Wildlife Serv., Fishery Bull., p. 95. JOHNSON, H. 1964. Human blood A1 specific agglutinin of the butter clam Saxidomus giganteus. Science, 146, 548-549. OYAMA, V. I., AND EAGLE, H. 1956. Measurement of cell growth in tissue culture with a phenol reagent ( Folin-Ciocalteau). PTOC. Sot. Exptk, BioE. Med., 91, 305307. SALTON, M. R. J. 1964. “The Bacterial Cell Wall,” 293 pp. Elsevier Co., New York. TRIPP, M. R. 1966. Hemagglutinin in the blood of the oyster Crassostrea uirginica. J. Invertebrate Pathol., 8, 478484. TRIPP, M. R., BISIGNANI, L. A., AND KENNY, M. T. 1966. Oyster amoebocytes in vitro. J. Znvertebrate Pathol., 8, 137-140. TYLER, A. 1946. Natural heteroagglutinins in the body fluids and seminal fluids of various invertebrates. Biol. Bull., 90, 213-219. WATKINS, WINIFRED M. 1966. Blood group substances. Science, 152, 172-181. WOODS, K. R., PAULSEN, E. C., ENGLE, R. L., AND PERT, J. H. 1958. Starch gel electrophoresis of some invertebrate sera. Science, 127, 519-520.