Characterization of a Natural Agglutinin Present in the Hemolymph of the California Sea Hare,

substance in the serum of the gastropod, Aplysia capable of agglutinating and vertebrate red blood cells was subjected to physicochemical analysis in order to ascertain its possible nature. Our studies indicate that agglutinating activity is due to a heterogeneous group of high molecular weight mole- cules with two activity peaks exhibiting sedimentation coefficients centering -18.5 S and -31.0 S. This material has a protein component associated with the active sit,e since it is sensitive to heat, pH extremes, and extraction with 2-mercaptoethanol, phenol, chloroform, and trichloracetic acid. Its physicochemical characteristics are different from other known invertebrate agglutinating substances and from classical vertebrate antibody.

Recently McKay et al. (1969) found that serum from Aplysia sp. agglutinated vertebrate red blood cells. A study was undertaken to determine whebher serum from the California sea hare, ApEysziz calijomica, contained bacterial agglutinins and to characterize the physicochemical properties of any agglutinin found. This paper reports the detailed characterization of a natural agglutinin in the serum of A. californica capable of reacting with bacteria and vertebrate red blood cells. A small portion of this study has appeared in an earlier communication (Pauley, 1971).

MATERIALS AND METHODS
Animal collection and maintenance. Animals for experimental use were collected intertidally throughout the year at the Irvine Horse Ranch south of the Game Preserve in Corona de1 Mar, southern California.
Animals were maintained in a 207 200gallon salt water aquarium cooled to 17 f 2°C. This aquarium was supplied with airstones and subsand filters covered by crushed oyster shells to maintain a slightly basic pH, and overlaid with washed activated charcoal. Water was added to the aquarium to replace evaporation loss and 25 gallons of water w as changed each mont'h. Salinity was monitored wit'11 a hydrometer. Animals were fed fresh algae (Egresiu sp.) twice a month; uneaten plants were immediately removed if t'hey started to decompose. Sea hares thrive extremely lvell under these condit,ions for 2-3 months.
Bacterial cultures. All the test bacteria were grown on seawater agar slants (Johnson, 1968 Johnson (1969). A fifth bacterium used in this study is a chromagenic gram-negative rod (Ap5Y) isolated by the senior author from the foot mucous of A. califomica. This organism forms smooth round, brilliant yellow colonies on seawater agar plates. Test cultures of all bacteria were grown for 48 hr except, for Ap5Y which was grown for only 24-36 hr because it grew more quickly than others. Bacteria used in our tests were washed off the slants wit,h sterile seawater.
Serum preparation. Whole hemolymph was withdrawn from the engorged pedal sinus of A. califomica using a sterile syringe fitted with a Z-gauge needle after cleaning the pedal surface with 70% ethanol. This procedure caused little observable trauma if animals were held in moist paper towels. Withdrawn fluid was quickly transferred to sterile test tubes placed in an ice bath. Fluid from different sea hares was not pooled except for pH activity and molecular weight studies. Cells were removed from the hemolymph by centrifuging the tubes at 2000 rpm/4 min. All centrifugations in this study were carried out at 4°C. The supernatant fluid was drawn off and passed through a Swinnex-25 Miliipore filter (0.45 P pore size) into sterile test tubes. If the filtered fluid \vas not immediately used, the tubes were frozen-stored at -12 or -18°C. Filter-sterilized hemolymph prepared in the above manner will be referred to as serum. For molecular weight studies, t.he serum was concentrated by pressure dialysis to remove water and small ions using Amicon Ultrafiltrat.ion Cells fitted with a PM 10 Diaflow Ultrafilter having a molecular weight exclusion limit of 10,000 (Amicon Corp).
Agglutination Assay. Test bacteria or red blood cells (RBCs) from chickens and pigs (obtained preserved in Alsevers Solution from Flow Laboratories) were washed t,hree times in eit.her sterile seawater or sterile 0.15 M saline. Bacteria were centrifuged at 3600 rpm/3 min and adjusted to a final concentration of 3.0 X log cells/ml by visual comparison with a barium sulfate nephelometer.
RBCs were centrifuged at 2000 rpm/5 min and adjusted to a 2.0% final concentrat,ion.
Serial 2-fold dilutions of 1.0 ml sea hare serum were made in Kahn tubes to which was added 0.05 ml of bacteria or RBCs. Dilutions were made with either sterile sea water (bacteria) or saline (RBCs).
Agglutination titers were read after 18-24 hr incubation at 26°C or after 36-48 hr incubation at' 13°C. We considered agglut,ination activity to be strong (+ + +) when all bacteria seemed to be agglutinated into numerous large clumps. In weak reactions (+), not all of the bacteria were agglutinated, although many small clumps were observed. When clumping did occur in control tubes, there were significantly fewer clumps than were found in weak reaction (+) tubes. Control and experimental tubes were always assayed side by side and only those experimental tubes exhibiting a marked difference were considered positive for agglutinating activity. The titer end point was defined as the highest serum dilution which permitted visible agglutination (+) as determined with a dissecting microscope at X30 and agglutinat'ing activity was expressed as the reciprocal of the titer dilution. Three controls were used in each of the experimental agglutinin tests: (1) control tubes cont,aining untreated sterile seawater; (2) control tubes containing sterile seawater which received the experimental treatment; and (3) control t,ubes containing untreated or dialyzed serum. Tests were performed in triplicate on the serum from each of five donors. These tests were repeated twice, usually against two different organisms. Agglutinin specificity was tested by incubating 1.0 ml serum overnight at 26°C with 7 X log bacteria and then assaying for agglutinating activity against the same organism, other bacteria strains, or RBCs. To determine whether "0" ant'igen is involved in the attachment site of the agglutinin, 0.2 ml of purified Difco E. coli "0" antigen was employed as a blocking antigen and incubated in a t,est tube with 0.5 ml of sea hare serum for 24 hr at 26°C. Physical tests of stability. The effect of lowered temperatures was studied by (1) storing serum at -12°C and testing agglutinin activity after 1, 2, 3, 4, 5, and 6 months, and (2) measuring agglutinin activity of serum aft'er repeated freezing and thawing as described by Krassner and Flory (1970). Heat stability was studied by exposing serum to one of the following temperatures for 20-30 min: 40", 50", 6O", 70", 80", and lOO"C, after which the serum was rapidly cooled by plunging the t,ube into an ice bath. Any formed precipitates were removed by cent,rifuging the fluid at 5000 rpm/20 tin; the wit,hdrawn supernat'ant was tested for agglutinating activit'y. Serum was dialyzed in 8 X 100 Visking tubing (Union Carbide Corporation) for 24-48 hr at 4°C against one of the following solutions: (1) 0.15 M NaCl (pH 6.0), (2) sterile seawater, or (3) 0.15 M NaCl, 0.01 M Tris-HCl buffer (pH 8.6). This last solution will be abbreviated as THB throughout the paper. Sera from a number of donors showed a pH range of 8.4-8.7, with a mean value of 8.6. These sera were then pooled and placed into a number of test tubes and the pH was adjusbed to various levels by adding either 1 M HCl, 10 M HCl, 1 M NaOH, or 10 M NaOH. After 24 hr incubation at 26"C, precipitat,es were removed by centrifugation at 5000 rpm/20 min and the supernatant dialyzed 24 hr against three changes of THB (pH S.6) at 4°C.
Chemical tests of stability. Test and control sera were extracted with phenol as described by Granger (1968, 1970) with the exception that dialysis was carried out against 0.15 M NaCl (pH 6.0). Diethyl-ether extractions were used as one type of control in these tests. Chloroform extractions were carried out as follows: chloroform, glass--are, and serum were precooled at 4°C and 3 pa& of chloroform were gently mixed with 1 part of serum in a separatory funnel for about 3 min. The chloroform phase containing precipitate was separat'ed from the aqueous (serum) phase and the latter centrifuged at 5000 rpm/3-4 min to remove excess precipitate. The serum phase was then dialyzed for 24 hr at 4°C against THB (pH 8.6). Toluene and xylene extractions were carried out in the same manner as chloroform exOraction except that 5 parts of toluene or xylene were mixed with 1 part of serum. Trichloroacetic acid (TCA) extraction was performed by incubating 0.5 ml serum in a test tube containing 0.5 ml 20% TCA for 2 hr at 4°C. Formed precipitate was removed by centrifuging at 3000 rpm/20 min, and t'he supernatant was dialyzed for 24 hr at 4°C against THB (pH S.6). Inactivation with 2-mercaptoethanol was done by precooling t'he chemical, sea hare serum, and glassware to 4°C. One milliliter of serum was gently mixed with 0.2 ml of 1 M P-mercapt,oethanol and incubated for 24 hr at 4°C. The mixture was then centrifuged at 3000 rpm/20 min to remove any precipitate and dialyzed 48 hr at 4°C against THB (pH 8.6). Sodium citrate chelation was carried out by dialyzing serum for 24 hr against 0.4 M sodium citrate (Acton et al., 1969), and then dialyzing against THB (pH 8.6) for 24 hr at 4°C. To det,ermine the effect of urea on agglutinin, an equal volume of serum and 8 M urea were gently mixed and incubated overnight at 26°C. The mixture was then dialyzed for 24 hr at 4°C against THB (pH 8.6).
Enzyme sensitivity tests. Five enzymes (Worthington Laboratories) were tested for their effect on agglutinin activity of sea hare serum. These were used at concentrations of 1.0 or 10.0 pg/ml serum. Enzymes were incubated wit,h sera overnight at the pH and temperatures indicat.ed: trypsin-pH 8.0, 37°C; pronase-pH 7.0, 37°C; neuraminidase-pH 5.1, 37°C; ribonuclease-pH 7.3, 37°C; and deoxyribonuclease-pH 7.0, 26°C. Precipitates were removed by centrifuging at 3000 rpm/5 min. As a cont,rol, proteolytic enzymes were checked for their activity against hemoglobin at the pH and temperatures listed. Hemoglobin was then eluted on Brinkman MN-polygram Cel300 to 20 X 20 cm precoated thin-layer chromat,ography (TLC) sheets, using an elut,ing solvent consist,ing of pyridine, isoamyl alcohol, and water in the ratio of 45: 35: 35.
Molecular weight studies. Molecular sieve column chromatography using various Sephadex gels n-as employed initially in an attempt to approximate the molecular weight of sea hare agglutinin. A slurry of Sephadex G-50 or G-100 was poured into a Siliclad (Clay-Adams) coated glass column, 1.3 cm in diameter, to a final height of 20 cm. Sephadex G-200 slurry n-as poured into a coated glass column, 1.6 cm in diameter to a final height of 26 cm. In all cases the columns were equilibrat'ed for 24 hr nit,h THB (pH 8.6). A sample (O.ci ml or 1.0 ml) of concentrated (10X) A. calijomica serum was loaded on the column and eluted with THB. The flow rate was adjusted to 1 m1/12.5 min. Two-milliliter fractions were collected and tested first for the amount of protein present by absorption at 280 nm in a Gilford 2000 spectrophotometer and then assayed for agglutinating activity. Subsequently, sucrose density gradients (lo-40 %) using ultracentrifugation were employed to ascertain the molecular weight of the agglutinin. Beckman cellulose nitrate tubes (1" X 3") were used to make 26-ml continuous sucrose gradients by diluting 40 % sucrose with THB (pH 8.6) on a Buchler polystaltic pump. The gradients were stored in a 4°C cold room overnight before use. Concentrated (5X) A. caZifornica serum and protein markers were carefully layered on the top of the gradient. Protein markers used were bovine serum albumin (mol. wt'. 67,000 or 4.4 S) and human y-globulin (mol. wt. 150,000 or 7 S). Samples were centrifuged for 6 hr in a Beckman L-2 ultracentrifuge at 22,000 rpm, using a SW 2.5.1 rotor. By use of the polyst'altic pump, 45% sucrose containing phenol red as a marker was layered beneath Dhe sucrose gradient, and 2-ml fractions were collected from the top of the tube as the lighter gradient was moved upward. Each fraction was tested for protein by absorption at 280 nm in a Gilford 2000 spectrophotometer, dialyzed 24 hr against THB (pH 8.6) at 4°C to remove the sucrose, and then assayed for agglutinating activit,y.

Agglutination. Titers
Normal serum agglut'ination titers of Aplysia calijornica varied between individuals although we never saw more than a 2-to 4-fold difference in activity (Table 1). All four marine bact'eria tested were agglut,inated, but Serratia nzarcescens, a terrestrial pathogen of insects, was not agglutinated. The absence of agglutination by sea hare serum in t'his case may be due to the fact that the capsule of S. marcescens masks bacterial cell wall ant*igens (Nowotny, 1969). Normal sea hare serum agglutinated both chicken and pig RBCs w&h very strong activity against chicken cells. A prozone sometimes aIt is noted that on occasion bacteria and chicken R.BCs clumped in t,he saline control tubes. In these cases, control and experimental tubes were assayed side by side and only those experimental tubes exhibiting a marked difference were considered positive for agglutinating activity.
noted in the most concentrated serum tubes, was especially frequent when assay was made using the gram-negative rod Ap5Y. The results of the cross agglutination tests show that any of the marine bacteria are capable of completely absorbing out of normal sea hare serum the agglutinin against itself, the other marine bact,eria, and the two RBCs, indicat'ing t'he agglutinin is nonspecific (Table 2). However, S. mat-ceseens is incapable of absorbing the agglutinin out of the serum, which is not surprising since it is not agglutinated by norma serum. Purified E. coli "0" ant'igen acts as a blocking agent which partially inactivates the agglutinin ( Table 2). The hemagglutinins in the snail Viviparus malleatus (Cheng and Sanders, 1962) and the oyster Crassosfrea virginica (Tripp, 1966) have been found to have agglutinating titers directly proportional to the total serum protein concentration. Therefore, we analyzed the total protein concentration in the serum of several -4. califomica by the Folin-Ciocalteau method described by Lowry et al. (1951). Normal serum protein concentration in the sea hares was found to vary among individual animals and may be inversely proportional to the agglutination titer (Table 3). range of pH values between 5 and 10 and 1). Reduction of titer n-as most pronounced below pH 5. The naturally occurring agglu- 6O"C/30 min but is completely inactivated after incubation at 7O"C/20 min (  The stability of the agglutinin to dialysis indicated a macromolecule was involved, and subsequent tests using molecular sieve column chromatography and sucrose gradient separations were employed in an attempt bo approximate the size of the agglutinin molecule. Sephadex separation indicates that the molecular weight is probably greater than 150,000 assuming it is a globular protein. The elution pattern on Sephadex G-50, G-100, and G-200 is the same as blue dextran which has a molecular weight of 2 X 106. An example of the separat'ion pattern in a G-200 column is shown in Fig. 2 where the agglut,inin appears in the single major protein fraction which is eluted with the blue dextran. After ultracentrifugation of A. californica serum in sucrose gradients, agglutination activity was found in all fract,ions conbaining sea hare protein (Fig. 3). Additionally, a secondary peak was observed in fractions 13 and 14 which contained some protein detectable by optical density measurement,s (Fig. 3). The major agglutination act,ivity appears to be a heterogeneous group of molecules with a sedimentation coefficient' centering around 18.5 S, while the minor peak has a sedimentation coefficient of approximately 31 S. These observaCions indicate that the agglutinin present in sea hare serum is a very large molecular weight material. The sedimentation coefficients of the two agglutinin peaks may be subject to some error, since we made the estimate based on the ultracentrifugation pattern of only t,wo protein markers. However, the sucrose density ult,racentrifugation studies do corroborate earlier findings that t#he agglutinin has a molecular weight greater t'han 150,000 as indicated by its elution pattern on a Sephadex G-200 column.

DISCUSSION
The insusceptibility of Aplysia californica agglutinin to RNase, DNase, and neuraminidase, in conjunction with it,s sensitivity to phenol and TCA extraction, suggest that its biological activity is not dependent upon nucleic acids or polysaccharides. Its sensitivity to TCA, phenol, and chloroform extraction indicate that the activity may be due to a protein or lipoprotein. Although lipoproteins are dissolved in phenol (Nowotny, 1969), a lipid component in Dhe agglutinin as outlined in the text. Collected fractions were assayed for agglutinating activity against M. aquivivus (628). Line graph shows the optical density of each fraction as measured at 280 nm. Agglutinating activity is indicated by bar graphs, Agglutinating titer of concentrated control serum not subjected to column chromatography was 32. Blue dextran was eluted in tubes 11 and 12, while phenol red was eluted in tubes 34-38.
was ruled out on the basis of several other that some proteins, such as mouse lymphotests. Toluene and xylene will inactivate toxin (LT), are extremely tolerant to exlipids (Iirassner and Flory, 1970), and most t,remes of both heat and pH (Iiolb and lipoproteins are easily denatured by diethyl Granger, 1970). Cleavage of cystine disulfide ether at temperatures as low as O'C (Scanu, bonds in proteins is accomplished with 1965), freezing, or repeated freezing and 2-mercaptoethanol (Fougereau andEdelthawing (Hatch andLees, 1968). However, man, 1965). The lack of sensitivity of the A. califomica agglutinin is not inactivated agglutinin to proteolytic enzymes indicat.es by any of these lipid and lipoprotein tests. that either the enzymes were not active or Current evidence, therefore, indicates that they do not. affect the active sit,e. The first of the agglutinin is protein or contains a major these possibilit.ies can be eliminat,ed because protein component. This assumption is based the enzymes were active as determined by on the agglutinin's susceptibility to heat, pH their effect on hemoglobin eluted on TLC extremes, and to extractions lvith 2-mercap-sheets. Granger (1968, 1970) obtoethanol, chloroform, phenol, andTCA. served that although both human and mouse Protein is precipitated by chloroform (un-LT were protein as determined by their published data), phenol (Palmer and Ger-bouyant densities, they were resistant to lough, 1940;Nowotny, 1969), and TCA trypsin digestion. This may be due to the (MacInnis and Voge, 1970). The inactivafact that trypsin acts upon specific sites tion of A. califomzica agglutinin by heat and which are not exposed or are not present in extremes of pH is characteristic of proteins LT molecules. Pronase, on the other hand, (Fox and Foster, 1957;Florkin and Botz, has no specific sit.e of action and if the agglu-1963). However, it. should be point,ed out tinin is proteinaceous it should therefore be profile of concentrated A. californica serum (AP) was subjected to a 10-40yo sucrose gradient as outlined in the text. Collected fractions were assayed for agglutinating activity against Pseudomonas sp. (FR). Line graph shows the optical density of each fraction as measured at 280 nm. Agglutinating activity is indicated by bar graphs. Concentrated control serum not subjected to ultracentrifugation had an agglutination titer of 128. Bovine serum albumin (BSA) was found to have a maximum optical density in tube 2, while the maximum optical density of human r-globulin (IgG) was in tube 3.
inactivated by this enzyme. The apparent protective complex (e.g., with a polysacchainsensitivity of the agglutinin to pronase is ride that protects the active site but is not unknown, but there are three possible expla-itself involved in the biological reaction of nations for this. It may be that pronase agglutination). Sea hare agglutinin was not affects the A. californica agglutinin nonspeaffected by urea, a compound which causes cifically only in a specific region of the moledissociation of hydrogen bonds in proteins cule, as does pepsin, which degrades only the and will often separate a protein into com-Fe fragment of vertebrate antibody into ponent subunits, as occurs in horseshoe crab small peptides (Fougereau and Edelman, hemagglutinin (Marchalonis and Edelman, 1965). Since the agglutinin molecule is ex-1968). Sodium citrate had no effect on the tremely large, it may be present as a complex agglutinin, indicating that it is not dependthat is not easily degraded by pronase. It is ent upon bivalent cations for stabilization. possible that pronase can cleave the agglu-The presence of disulfide bonds in vertebrate tinin into subunits, but is not capable of act-antibody may be detected by the use of ing on the subunits due to the formation of a 2-mercaptoethanol (Fougereau and Edelman,  PAULEY,  GR$NGER,  AND  KRASSNER 1965), and the pa&al inactivation of sea hare agglutinin by this chemical indicates that disulfide bonds are present. Incomplete inactivation with t,his chemical may be because alkylation with iodoacetamide was not performed, thereby permitting the disulfide bonds to recombine under the alkaline conditions of the experiment] to yield some agglutinin-active sites. The possibility also exists that the molecule is coiled in such a way as to prevent complete unwinding upon breaking of the disulfide bonds, thereby partially preserving the integrity of the active s&e.
The partial inactivation of the agglutinin by E. coli "0" antigen indicates that' the attachment site of the agglutinin is, at least in part, a polysaccharide similar in structure to "0" antigen. This in part helps to explain the inability of sea hare serum to agglutinate S. marcescens, since the "0" antigens are protected by a capsule in this bacterium (Nowotny, 1969). Of the few invertebrate agglutinins that have been characterized in detail, oyst,er hemagglutinin appears to be most similar t,o sea hare agglutinin. C. virginica hemagglutinin is unaffected by dialysis, although aging renders it dialyzable; it exhibits cross reactivity; it is heat, labile; and it is a protein wit'h an extremely large sedimentation coefficient of 33.4 S (Tripp, 1966;Li and Flemming, 1967;Acton et al., 1969). Oyster hemagglutinin differs from sea hare agglutinin in that it is stable over a narrower pH range (pH 6-9) and breaks down into subunit.s beyond pH 7-5 (Li and Fiemming, 1967;Acton et al., 1969). However, the reduced titer of sea hare agglut'inin at pH extremes may indeed indicate dissociation of the molecule into subunits. Another difference between the two agglutinins is that oyster hemagglut8ination activity is proportional to serum protein levels (Tripp, 1966), whereas an inverse correlation is found between sea hare agglutinin and serum protein concentration.
However, more data are needed before any definit.ive statement can be made. Li and E'lemming (1967) found oyster hemagglutinin aet.ivity associated with two distinct protein peaks after separation by Sephadex G-75 columns, indicating a molecular weight less t,han 7.',000. This is in fair agreement \vit,h McDade and Tripp (1967)) who estimat,ed a molecular weight above 65,000. These authors were probably working with the subunits rat,her t,han the intact molecule because ActIon et, al. (1969) has since shown that oyster hemagglut#inin is composed of noncovalently linked subunits, wit.h a molecular weight of 20,000, and t.hat t,he entire molecule with all its subunits intact has a very high sedimentation coefficient of 33.4 S. This is fairly close to t,he minor 31 S activit,y peak of sea hare agglutinin. Two studies have shown t,hat, oyster hemagglutinin is stabilized by calcium ions (McDade and Tripp, 1967;Acton et, al., 1969), but this is not true for A. calijordca agglutinin since it, was not inactivated by sodium citrate.
A hemagglutinin present in t,he mussel VelesuGo am&&us has recent,ly been purified and characterized (Jenkin and Rowley, 1970). The material was precipitated by 50 % saturated ammonium sulfate, and subsequently purified by sucrose densit'y gradient centrifugation.
Its sedimentation coefficient of 28 S is similar to the minor 31 S activit(y peak of sea hare serum. The mussel hemagglutinin was capable of being cross absorbed with different vertebrate RBCs, as was sea hare agglut<inin. Purified mussel hemagglut.inin was found to be protein and subsequent amino acid analysis revealed no sulfur-containing amino acids, differing from sea hare agglutinin which has disulfide bonds. Horseshoe crab (Limulus polyphemus) hemagglutinin has been studied extensively by Marchalonis and Edelman (196S), who estimated a molecular weight of about 400,000 for this molecule, which like oyster hemagglutinin could be separated into subunits of 22,500 molecular weight by exposure t,o pH 3.0 or 9.6 followed by treatment ~.ii h S.0 M urea. They also showed that horseshoe tion of the sea hare agglut'inin and this Jvill be crab hemagglutinin was stabilized by caldiscussed in detail in a subsequent paper cium ions and did not have covalent bonds, concerning in vivo bacterial clearance in such as disulfide bonds, between the sub-t,hese gastropods. units. Cornick and Stewart (19GSa) found that the bacterial agglutinin in another ACKNOWLEDGMENTS -arthropod (Homarus americanus) was un- C. R., AND ROWLEY, D. 1970. Immunity in invertebrates.
The purification of a haemagglutinin to rat and rabbit erythrocytes from the haemolymph of the murray mussel (Velesunio