Purification and characterization of a natural agglutinin from the serum of the hermit crab Diogenes affinis

Biochimica et Biophysica Acta 1472 (1999) 13^24 www.elsevier.com/locate/bba

Puri¢cation and characterization of a natural agglutinin from the serum of the hermit crab Diogenes a¤nis Sathasivam Murali, Periasamy Mullainadhan *, Munusamy Arumugam Laboratory of Pathobiology, Department of Zoology, University of Madras, Guindy Campus, Madras 600 025, India Received 20 January 1998; received in revised form 2 June 1999; accepted 3 June 1999

Abstract A natural agglutinin from the serum of the hermit crab Diogenes affinis was purified to homogeneity by a single-step affinity chromatography using N-acetylglucosamine-coupled Sepharose 6B. The purified serum agglutinin (PSA) showed a strong affinity for rat RBC, and its hemagglutinating (HA) activity was specifically dependent on Ca2‡ and reversibly sensitive to EDTA. PSA in active form has a molecular mass estimate of 185 kDa and is composed of four non-identical subunits (51, 49, 42 and 39 kDa) cross-linked by interchain disulfide bonds. The homogeneity of PSA was corroborated by immunodiffusion and immunoelectrophoretic analyses using rabbit antiserum raised against the agglutinin. The antibodies in this antiserum appear to be specific for RBC-binding sites of the agglutinin molecules as revealed by the ability of the antiserum to neutralize HA activities of both whole serum and PSA of D. affinis. In HA-inhibition assays performed with several carbohydrates and glycoproteins, PSA showed a distinct and unique specificity for acetyl group in carbohydrates independently of the presence of this group on C-2 or C-5 and its stereochemical arrangement in the axial or equatorial orientation. Besides, this agglutinin appears to recognize the terminal N- and O- acetyl groups in the oligosaccharide chain of glycoconjugates. The HA activity of D. affinis agglutinin was also susceptible to inhibition by lipopolysaccharides from diverse Gram-negative bacteria, which might indicate a significant in vivo role of this humoral agglutinin in the host immune response against bacterial infections. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Invertebrate immunity ; Crustacean; Serum agglutinin; Puri¢cation; Acetyl group speci¢city; Bacterial lipopolysaccharide binding activity

1. Introduction In invertebrates, agglutinins have been detected not only in the blood components, namely cell-free hemolymph and hemocytes [1^5], but also in tissues [6,7], and mucus [8]. Although the actual functions of invertebrate agglutinins are not yet well understood, they have been, however, implicated in several di-

* Corresponding author. Fax: +91-44-235-2494; E-mail: [email protected]

verse physiological processes, including host immune responses, such as non-self recognition, phagocytosis, encapsulation, and hemocoelic clearance of foreign cells [3,9^13]. Besides, some of the invertebrate agglutinins, with de¢ned speci¢city for simple or complex carbohydrate conjugates, have been used as invaluable tools in a variety of biomedical applications [14^18]. In seeking an explanation for the functional properties of agglutinins in homologous or heterologous systems, emphasis is always placed on their ability to recognize speci¢c carbohydrate structures [12,19]. In-

0304-4165 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 9 9 ) 0 0 0 9 7 - 5

BBAGEN 24851 30-9-99

14

S. Murali et al. / Biochimica et Biophysica Acta 1472 (1999) 13^24

vertebrate agglutinins, for example, are known to speci¢cally recognize the whole sugar [20], a speci¢c part of a sugar [21], a sequence of sugars [22], or their glycosidic linkages [23]. Invertebrates are exceedingly diverse which comprise a number of animal groups often with polyphyletic origin, and it is, therefore, plausible that novel agglutinins having unique carbohydrate binding speci¢cities and biological activities will be found from these organisms. Since marine invertebrates, particularly crustaceans, are rich sources of agglutinins with a¤nity for a variety of carbohydrates [5,24,25], we have screened sera from several species of marine crustaceans for the presence of agglutinins with unique carbohydrate binding speci¢cities. Recently, we reported the occurrence of a natural agglutinin with an apparent speci¢city for acetylated amino sugars in the serum of the hermit crab Diogenes a¤nis [26]. In this paper, we describe the serological and chemical characteristics, carbohydrate-binding speci¢city and bacterial lipopolysaccharide-binding property of this novel agglutinin puri¢ed from the serum of the hermit crab.

bay, India) or Sigma. Freund's complete adjuvant was purchased from Difco (Detroit, MI, USA). All other chemicals and reagents used were of the highest analytical grade commercially available. Five types of Tris-bu¡ered saline (TBS) were used: TBS-I, 50 mM Tris-HCl, 135 mM NaCl (300 mOsm); TBS-II, 50 mM Tris-HCl, 115 mM NaCl, 10 mM CaCl2 (300 mOsm); TBS-III, 50 mM TrisHCl, 35 mM NaCl, 10 mM CaCl2 (135 mOsm); TBS-IV, 50 mM Tris-HCl, 100 mM NaCl, 50 mM EDTA (300 mOsm); and TBS-V, 50 mM Tris-HCl, 1 M NaCl, 10 mM CaCl2 (2070 mOsm). All the bu¡er solutions were adjusted to pH 7.5 and contained 0.02% NaN3 .

2. Materials and methods

2.3. Preparation of crab sera

2.1. Reagents and bu¡ers

Hermit crabs D. a¤nis weighing 8^15 g (without shell), irrespective of sex, were bled from the cut end of the dactylus region of the leg. Serum was obtained from pooled hemolymph samples as described elsewhere [26], and used immediately.

Epoxy-activated Sepharose 6B was purchased from Pharmacia (Uppsala, Sweden). Carbohydrates were products of BDH (Bombay, India), Fluka (Basel, Switzerland), Merck (Darmstadt, Germany), Serva (Heidelberg, Germany), or Sigma (St. Louis, MO, USA). Glycoproteins, standard molecular weight marker proteins (SDS-6H) for gel electrophoresis and bacterial lipopolysaccharides (LPS) were purchased from Sigma. The marker proteins for HPLC were from Tosoh (Tokyo, Japan). De-O-acetylation of bovine submaxillary mucin was performed following the procedure of Sarris and Palade [27]. The types of LPS used include those from Salmonella abortus equi (phenolic extraction (PE)), Klebsiella pneumoniae (PE), Escherichia coli 0111:B4 (PE), Serratia marcescens (PE), Pseudomonas aeruginosa serotype 10 (PE) and Salmonella minnesota R5 (Rc mutant: phenol-chloroform-petroleum ether extraction). Amino acids were products of SRL (Bom-

2.2. Erythrocytes (RBC) Human and other blood samples from diverse mammalian species were obtained by venous or cardiac puncture and collected in sterile Alsever's solution [28] containing 100 Wg/ml of streptomycin. RBC were washed three times with 0.9% saline and once with TBS-II and ¢nally resuspended in TBS-II as 1.5% suspension (v/v).

2.4. Preparation of GlcNAc-Sepharose a¤nity column N-Acetylglucosamine (GlcNAc) was immobilized on Sepharose 6B following the procedures of Vretblad [29] and Osterman [30]. Brie£y, epoxy-activated Sepharose 6B (1 ml) was washed extensively with double distilled water and 0.1 M NaOH. The gel was mixed immediately with 1.5 ml of 0.1 M NaOH containing 75 mg GlcNAc and the suspension was incubated with gentle mixing at 45³C for 15 min. The gel was then washed extensively with 0.1 M borate bu¡er (pH 8), 0.1 M acetate bu¡er (pH 4), both containing 0.5 M NaCl, and with 0.9% saline. The gel was ¢nally equilibrated with TBS-II and transferred to a 0.8U6.5 cm polypropylene column.

BBAGEN 24851 30-9-99

S. Murali et al. / Biochimica et Biophysica Acta 1472 (1999) 13^24

2.5. Puri¢cation of serum agglutinin Serum diluted with TBS-II (95^100 ml) to a hemagglutination (HA) titer of 16 against rat RBC was passed through the a¤nity column. The unbound serum components were removed from the column by washing with a low salt bu¡er (TBS-II) until the absorbance of the e¥uent at 280 nm almost returned to zero. Subsequently, the column was washed with a high salt bu¡er (TBS-V) until the A280 of the e¥uent reached zero and then re-equilibrated with TBS-II. The agglutinin bound to the a¤nity matrix was eluted with 0.2 M GlcNAc in TBS-III and the fractions which showed absorbance at 280 nm were collected and dialyzed (MW exclusion limit 6 10 000) extensively against TBS-II at 10³C to remove GlcNAc. If necessary, the fractions containing HA activity were pooled, lyophilized and stored at 35³C until use. All the chromatographic procedures were performed at a steady £ow rate of 7 ml/h at 23³C, and the e¥uents (0.5 ml fractions) collected during adsorption, washing, re-equilibration and elution were tested for HA activity using rat RBC. 2.6. HA assays HA assays were performed in V-bottom microtiter plates (Greiner, Nu«rtingen, Germany) by serial dilution of a 25-Wl sample with an equal volume of TBSII. RBC suspension (25 Wl) was added to each well, mixed, and incubated for 45 min at 26³C. The HA titers were recorded as the reciprocal of the highest dilution of the sample causing complete agglutination of RBC. Control for all assays consisted of the substitution of the sample by TBS-II. Each experiment was performed in duplicate with at least ¢ve samples and the HA activities were analyzed based on the median HA titer values. Tests for requirement of divalent cations for HA activity and EDTA sensitivity of puri¢ed serum agglutinin (PSA) were carried out as described previously [26]. 2.7. HA-inhibition assays PSA sample was diluted with TBS-II to a HA titer of 8 against rat RBC. The substances to be tested for inhibition were dissolved in TBS-III (carbohydrates

15

and amino acids) or in TBS-II (glycoproteins and LPS). If necessary, the pH was adjusted to 7.5 using concentrated NaOH solution. The substance to be tested (25 Wl) was serially diluted with an equal volume of dilute PSA solution in microtiter plates and incubated for 1 h at 26³C. Rat RBC suspension (25 Wl) was added to each well and incubated for 45 min at 26³C. The minimal concentration of the test substance that completely inhibited the HA activity was recorded. 2.8. Protein determination Protein concentration was determined following the method of Lowry et al. [31] using bovine serum albumin as the standard. 2.9. Polyacrylamide gel electrophoresis (PAGE) The protein pro¢les of whole serum and PSA were analyzed in discontinuous PAGE under non-denaturing conditions (native PAGE) using a 3% stacking gel (pH 6.7) and a 7.5% separating gel (pH 8.9) in Tris-glycine bu¡er (pH 8.3) following Maurer [32]. Electrophoresis was run at a constant current of 4 mA per sample and at 10³C in a slab gel measuring 170U150U1.5 mm. The gels were stained with Coomassie Brilliant Blue R-250 [32], or with silver nitrate [33]. SDS-PAGE of PSA was carried out by the method of Laemmli [34] using a 3% stacking gel (pH 6.8) and a 10% separating gel (pH 8.8) in Tris-glycine bu¡er (pH 8.3). Prior to SDS electrophoresis, lyophilized PSA (120 Wg protein) was dissolved in 50 Wl sample bu¡er (0.06 M Tris-HCl bu¡er, 4% SDS, pH 6.8) that did or did not contain 5% L-mercaptoethanol. A mixture of standard molecular weight marker proteins (SDS-6H; 9 Wg protein) was also prepared identically. All samples were heated (10 min, 100³C), cooled to room temperature and electrophoresed (5 mA per sample, 20³C in a slab gel measuring 90U90U0.7 mm. The proteins were stained with Coomassie Brilliant Blue R-250. 2.10. Gel ¢ltration Native molecular-mass of PSA was estimated by gel ¢ltration in HPLC system (Pharmacia LKB,

BBAGEN 24851 30-9-99

16

S. Murali et al. / Biochimica et Biophysica Acta 1472 (1999) 13^24

Uppsala, Sweden) using a TSK G 3000 SWXL column (7.8 mm i.d.U300 mm; Tosoh, Tokyo, Japan), equilibrated with TBS-II at a £ow rate of 1 ml/min. The column was calibrated with reference proteins for molecular weight estimation under identical conditions. 2.11. Immunization, immunodi¡usion and immunoelectrophoresis PSA (0.5 ml containing 125 Wg protein per injection) emulsi¢ed with an equal volume of Freund's complete adjuvant was injected intramuscularly into the thigh of a male albino rabbit weighing approximately 1 kg. After 4 weeks, a booster injection of similar dosage, but prepared in Freund's incomplete adjuvant (liquid para¤n), was given as described above. Thereafter, this injection protocol was repeated every week for 4 weeks. Three days after the last injection, the rabbit was bled from the marginal ear vein and the freshly drawn blood was allowed to clot (12 h, 10³C). Antiserum was separated from the clots by centrifugation (400Ug, 5 min, 4³C), aliquoted and stored at 10³C with few crystals of merthiolate. The reactivity of the antiserum was determined by double immunodi¡usion (ID) in 1% agarose gels prepared in 25 mM borate-bu¡ered saline (pH 8.5) following Ouchterlony [35]. In all ID analyses, the samples of both preimmune serum and antiserum from rabbit were six-fold diluted with TBS-II, and 15 Wl sample to be tested was applied to each well. The gels were then incubated at 26³C for 48 h to develop the precipitation lines and stained with 6% amido black. Immunoelectrophoresis (IEP) was also performed

in 1% agarose gels in 116 mM barbital bu¡er (pH 8.6) following Garvey et al. [28]. Brie£y, 15 Wl of sample (whole serum or PSA) was applied to each well and electrophoresed at 70 mA for 2 h at 10³C. The antiserum (6-fold diluted; 150 Wl) was applied to the trough and the gel was incubated at 26³C for 48 h. The immunogenic protein bands were visualized by staining with 6% amido black. 2.12. E¡ect of antiserum on HA activity The ability of the rabbit antiserum raised against PSA to inhibit the HA activity of the whole serum or PSA of D. a¤nis was tested following Suzuki and Natori [36] with suitable modi¢cations. In this test, 25 Wl of undiluted preimmune serum or antiserum was serially diluted with an equal volume of TBS-II in microtiter plates. A 25 Wl amount of test sample (whole serum or PSA), previously diluted with TBSII to a HA titer of 8 against rat RBC, was added to each well. After mixing, the plate was incubated for 45 min at 26³C, and then 25 Wl of rat RBC suspension was added to each well. The inhibition of HA activity of the test samples was recorded after incubation for 1 h at 26³C. 3. Results 3.1. Puri¢cation of serum agglutinin Based on our preliminary HA-inhibition results demonstrating an apparent speci¢city of serum agglutinin of D. a¤nis for acetylated aminosugars [26], puri¢cation was attempted by a¤nity chromatogra-

Table 1 Summary of puri¢cation of agglutinin from the serum of D. a¤nisa Sample

Volume (ml)

Hemagglutination titerb

Total protein (mg)

Total activity (HA units)c

Speci¢c activity (HA units/mg protein)

Puri¢cation (-fold)

Serum (8-fold diluted) GlcNAc-eluate from a¤nity chromatography on GlcNAcSepharose 6B

97 3

16 512

1612 0.411

6.2U104 6.1U104

38 1.5U105

1 3900

a

Data represent the mean values of 15 separate experiments. Median titer values. c One unit of activity is de¢ned as the minimum amount of protein required to give one well agglutination of rat RBC. b

BBAGEN 24851 30-9-99

S. Murali et al. / Biochimica et Biophysica Acta 1472 (1999) 13^24

17

3.2. HA assays

Fig. 1. A¤nity chromatography of D. a¤nis serum agglutinin on a column of GlcNAc-Sepharose 6B. The dilute serum (97 ml) was applied to the column and sequentially washed with Tris-HCl bu¡er (TB) containing low (TBS-II) and high (TBS-V) NaCl concentration. After re-equilibration with TBS-II, the agglutinin was eluted with 0.2 M GlcNAc. Fractions of 0.5 ml were collected and assayed for HA activity using rat RBC.

phy using GlcNAc-Sepharose 6B. For a¤nity puri¢cation, the strategies for adsorption and elution steps were developed after optimizing pH, temperature, calcium ion and ionic strength dependence by batch methods [37]. A typical column pro¢le depicting the puri¢cation of D. a¤nis agglutinin on GlcNAcSepharose 6B is shown in Fig. 1. Both the dilute serum (about 1600 mg protein; HA titer = 16) which had passed through the a¤nity matrix and the e¥uent collected during subsequent washing of this matrix with TBS-II did not contain detectable level of HA activity against rat RBC. This indicates that all HA activity in the serum was adsorbed by the a¤nity matrix. Upon washing the column with high salt bu¡er (TBS-V), a small peak containing ca. 100 Wg inert proteins was eluted and this step was necessary to obtain the agglutinin in a homogeneous state. When 0.2 M GlcNAc was passed through the column, a sharp symmetrical peak of 280 nm absorbance as well as a coincident peak of agglutinating activity against rat RBC emerged from the a¤nity matrix. As summarized in Table 1, this chromatographic procedure consistently resulted in 3900-fold increase in speci¢c activity and 98% recovery of the total HA activity from the starting serum sample.

PSA reacted with all the 11 mammalian RBC types tested, namely, rat (titer 128), human A (32), B (32), and O (32), sheep (16), rabbit (16), mouse (160), goat (8), bu¡alo (8), horse (2) and ox (2) RBC types. The highest HA titer was obtained with rat RBC, and human A, B, and O RBC types were not discriminated by this agglutinin. In a separate experiment, the HA activity of PSA, dialyzed (MW exclusion limit 6 10 000) previously against divalent cation-free bu¡er (TBS-I) and then tested in the same bu¡er, decreased signi¢cantly from 128 to 8 against rat RBC. But this agglutinin completely recovered its original HA activity only upon the addition of Ca2‡ , but not Mg2‡ or Mn2‡ , to the reaction mixture, thereby indicating that this agglutinin speci¢cally requires Ca2‡ for its HA activity. Supportingly, the activity of PSA remained una¡ected after extensive dialysis against TBS containing Ca2‡ (TBSII). In another experiment (data not shown), a signi¢cant reduction in HA activity was noticed with PSA after treatment with 50 mM EDTA (TBS-IV)

Fig. 2. Electrophoretic analysis of PSA of D. a¤nis. (A) Native PAGE of whole serum (150 Wg protein, lane 1) and PSA (10 Wg protein) before (lane 2) and after (lane 3) adsorption (5U) with ¢xed rat RBC. (B) SDS-PAGE of PSA (lane 1) and MW marker proteins (lane 2) under reducing conditions. The protein bands in both gels were stained with Coomassie Blue.

BBAGEN 24851 30-9-99

18

S. Murali et al. / Biochimica et Biophysica Acta 1472 (1999) 13^24

and then tested in the absence of divalent cations. However, the addition of Ca2‡ , but not Mg2‡ or Mn2‡ , to this sample restored the original level of HA activity. 3.3. Electrophoretic and HPLC analyses In native PAGE (Fig. 2A), PSA of D. a¤nis gave a single protein band on staining the gel with Coomassie Blue (lane 2), and this band completely disappeared after adsorption of the sample with ¢xed rat RBC (lane 3). Identical results were obtained upon staining the gel with silver nitrate (not shown). The electrophoretic mobility of PSA seemed to correspond to a faint protein band often visible in the concurrent electrophoresis of the dilute whole serum (lane 1) of D. a¤nis. Under non-reducing conditions in SDS-PAGE, PSA appeared to dissociate, but this dissociation was found to be highly inconsistent when repeated several times under identical conditions. However, this agglutinin upon reduction with L-mercaptoethanol consistently dissociated into four bands whose molecular weights were 51, 49, 42 and 39 kDa (Fig. 2B; lane 1). With gel ¢ltration by HPLC, a symmetrical peak was observed for PSA in active form, and its molecular-mass was estimated to be 185 kDa (Fig. 3). 3.4. Immunological analysis In ID analysis, both PSA and the whole serum formed a single clear precipitation line in each case

Fig. 3. Molecular-mass determination of D. a¤nis PSA in active form (20 Wg; HA titer = 16) by gel ¢ltration on a TSK G 3000 SWXL HPLC column. Arrows show retention times of molecular-mass standards: 1, thyroglobulin (669 kDa); 2, Q-globulin (150 kDa); 3, ovalbumin (43 kDa); 4, ribonuclease-A (13.7 kDa); 5, p-aminobenzoic acid (0.137 kDa).

Fig. 4. Double immunodi¡usion (A) and immunoelectrophoretic (B) analyses of PSA (20 Wg protein, well 1) and whole serum (450 Wg protein, well 2) of D. a¤nis. Well 3 and trough contain rabbit antiserum raised against PSA. (C) Inhibition of HA activity of whole serum and PSA of D. a¤nis by rabbit antiserum raised against PSA. Serially diluted preimmune serum (row a) or antiserum from rabbit (row b) was incubated with whole serum. Similarly, the preimmune serum (row c) or the antiserum (row d), after serial dilution, was incubated with PSA. The pattern of HA activity against rat RBC is shown.

with the rabbit antiserum raised against PSA (Fig. 4A), and the preimmune serum did not show any reactivity (results not shown). The precipitation lines showed a converging arc of a homologous reaction, indicating complete identity of agglutinin molecules in the serum and the puri¢ed fraction in reactions with the antiserum. In IEP, PSA as well as the whole serum gave a single precipitation line with the antiserum and these lines identically migrated towards anode (Fig. 4B). Furthermore, the HA activity of both the whole serum and PSA of D. a¤nis was

BBAGEN 24851 30-9-99

S. Murali et al. / Biochimica et Biophysica Acta 1472 (1999) 13^24

19

Table 2 Inhibition of HA activity (titer = 8) of agglutinin (PSA) isolated from the serum of D. a¤nis by various carbohydrates Maximum concentration tested (mM)

Minimum inhibitory concentrationa (mM)

Hexoses Glucose Galactose Mannose

200.0 200.0 200.0

^b ^ ^

Hexosamines Glucosamine Galactosamine Mannosamine

200.0 200.0 200.0

^ ^ ^

N-Acetyl hexosamines N-Acetylglucosamine (GlcNAc) N,NP-Diacetylchitobiose (GlcNAc)2 N,NP,NQ-Triacetylchitotriose (GlcNAc)3 N-Acetylgalactosamine (GalNAc) N-Acetylmannosamine (ManNAc)

200.0 50.0 50.0 200.0 200.0

1.56 1.56 1.56 3.12 1.56

10.0 12.5

2.50 ^

Carbohydrates tested

Sialic acids N-Acetylneuraminic acid (NeuAC) N-Glycolylneuraminic acid (NeuGc)

The following carbohydrates also did not inhibit the HA activity and unless otherwise stated, all carbohydrates were tested at concentrations up to 200 mM: L-D-allose, D-arabinose, L-arabinose, cellobiose, 2-deoxy-D-galactose, 2-deoxy-D-glucose, dextran T70 (10 mg/ ml), dextran T500 (10 mg/ml), D-fructose, D-fucose, L-fucose, D-galacturonic acid, gentiobiose, D-glucuronic acid, lactose, laminarin (10 mg/ml), maltose, maltotriose, melezitose, melibiose, methyl-K-D-galactopyranoside, 1-O-methyl-L-D-galactopyranoside, methyl-K-Dglucopyranoside, methyl-L-D-glucopyranoside, methyl-K-D-mannopyranoside, p-nitrophenyl-K-D-galactopyranoside, p-nitrophenyl-L-Dgalactopyranoside (50 mM), p-nitrophenyl-K-D-glucopyranoside (50 mM), p-nitrophenyl-L-D-glucopyranoside (100 mM), palatinose, ra¤nose, L-rhamnose, L-sorbose, sucrose, trehalose, turanose, D-xylose. a The assay was repeated ¢ve times for each carbohydrate with identical results. b ^, no inhibition.

Table 3 Inhibition of HA activity (titer = 8) of agglutinin (PSA) isolated from the serum of D. a¤nis by amino acids and their derivatives Amino acids tested

Maximum concentration tested (mM)

Minimum inhibitory concentrationa (mM)

L-Leucine

200 200 200 200 200 200 200 200 200

^b 6.2 ^ 12.5 ^ 25.0 50.0 ^ 100.0

N-Acetyl-L-leucine L-Glutamine N-Acetylglutamine Hydroxy-L-proline O-Acetylhydroxy-L-proline N-Acetylhydroxy-L-proline Glycine N-Acetylglycine

The following amino acids also did not inhibit the HA activity at concentrations tested up to 200 mM: D,L-alanine, L-arginine, D,L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-histidine, D,L-isoleucine, L-lysine, L-methionine, D,L-phenylalanine, L-proline, D,L-serine, D,L-threonine, D,L-tryptophan, L-tyrosine, D,L-valine. a The assay was repeated six times for each amino acid with identical results. b ^, no inhibition.

BBAGEN 24851 30-9-99

20

S. Murali et al. / Biochimica et Biophysica Acta 1472 (1999) 13^24

Table 4 Inhibition of HA activity (titer = 8) of agglutinin (PSA) isolated from the serum of D. a¤nis by glycoproteins and bacterial lipopolysaccharides Substances tested Glycoproteins Bovine submaxillary mucin (BSM) Asialo-BSM De-O-acetylated BSM Fetuin Asialo-fetuin Porcine thyroglobulin Porcine stomach mucin Bacterial lipopolysaccharides Salmonella abortus equi Klebsiella pneumoniae Escherichia coli 0111:B4 Serratia marcescens Pseudomonas aeruginosa (serotype 10) Salmonella minnesota R5 (Rc mutant) a b

Maximum concentration tested (mg/ml)

Minimum inhibitory concentrationa (mg/ml)

10 5 5 10 5 10 10

1.25 2.50 2.50 2.50 ^b ^ ^

2 2 2 2 2 2

0.06 0.25 0.50 1.00 1.00 ^

The assay was repeated ¢ve times for each test substance with identical results. ^, no inhibition.

inhibited by the antiserum up to 6 wells corresponding to a dilution of 64 (Fig. 4C; rows b and d). The preimmune rabbit serum had no e¡ect on the HA activity of both these test samples (Fig. 4C; rows a and c). 3.5. HA-inhibition assays The binding speci¢city of D. a¤nis agglutinin was examined by HA-inhibition assays using several carbohydrates, amino acids, glycoproteins, and bacterial LPS. Of the 50 carbohydrates tested, only six inhibited the agglutinating activity of PSA against rat RBC (Table 2). Neither the simple hexoses nor their amino derivatives tested inhibited the HA activity at concentrations up to 200 mM. On the other hand, only their N-acetyl derivatives, namely, N-acetylglucosamine (GlcNAc), N,NP-diacetylchitobiose ((GlcNAc)2 ), N,NP,NQ-triacetylchitotriose ((GlcNAc)3 ), N-acetylgalactosamine (GalNAc), and N-acetylmannosamine (ManNAc) at 1.56 or 3.12 mM inhibited the HA activity of PSA. Similarly, N-acetylneuraminic acid (NeuAc) also inhibited (2.50 mM) the activity, whereas N-glycolylneuraminic acid (NeuGc) failed to inhibit even at a concentration of 12.50 mM. Among amino acids and

their derivatives tested (Table 3), only acetylated amino acids were inhibitory at concentrations ranging from 6.2 to 100.0 mM. In tests with sialo- and asialo-glycoproteins (Table 4), only BSM, asialoBSM, de-O-acetylated BSM, and fetuin inhibited (1.25 or 2.50 mg/ml) agglutination of rat RBC by PSA. Besides, the HA activity of PSA could also be inhibited (0.06 to 1.00 mg/ml) by ¢ve of the six bacterial LPS types tested (Table 4). 4. Discussion In this study, a naturally occurring agglutinin from the serum of D. a¤nis was successfully isolated at high purity by a¤nity chromatography using GlcNAc-Sepharose 6B as a¤nity matrix and 0.2 M GlcNAc for elution. The serological properties of PSA, as revealed by the pattern of its reactivity against various mammalian RBC types, were essentially the same as that of the whole serum [26], thereby indicating that the isolated fraction is responsible for all types of HA activity detected in the serum of D. a¤nis. This agglutinin appears to be a C-type agglutinin as it requires Ca2‡ for its HA activity [38] and it is reversibly sensitive to EDTA. Both

BBAGEN 24851 30-9-99

S. Murali et al. / Biochimica et Biophysica Acta 1472 (1999) 13^24

the whole serum and PSA gave the highest HA titer for rat RBC, and the minimal concentration of PSA required for complete agglutination of this RBC type was 6 ng/ml. Using this value and a typical HA titer value of 128 for whole serum against rat RBC, the hemolymphatic concentration of agglutinin in D. af¢nis was calculated to be 0.76 Wg/ml. In native PAGE, PSA gave a single protein band, and the disappearance of this band after adsorption of the sample with ¢xed rat RBC clearly demonstrates that the HA activity in the serum of D. a¤nis is associated with a single protein fraction, and that it could be puri¢ed to homogeneity by a single-step a¤nity chromatography on GlcNAc-Sepharose 6B. This agglutinin, with an estimated native molecular weight of 185 kDa, is composed of four non-identical subunits cross-linked by interchain disul¢de bonds. The appearance of a single clear precipitation line in gels for PSA of D. a¤nis in ID and IEP analyses provides further evidence for purity of the isolated agglutinin against which the antiserum was raised. Similar results obtained with the whole serum against this antiserum demonstrate that the agglutinating molecules in the serum are antigenically distinct from other serum proteins of D. a¤nis. In addition, the puri¢ed molecules with agglutinating activity are antigenically identical to those present in the whole serum, as revealed by a complete identity observed in the reactivity of these two test samples with the antiserum in ID analysis. Moreover, the ability of this antiserum to neutralize the HA activity of PSA as well as the whole serum of D. a¤nis show that antibodies in the antiserum are apparently speci¢c for RBC-binding sites of the agglutinin molecules. Serum agglutinins in some crustaceans, namely, the lobster [39], prawns [40^43], and crabs [44,45], appeared to be speci¢c for acetylated aminosugars, but these agglutinins: (a) did not recognize NeuAc; (b) were not tested for their reactivity with appropriate hexosamines; or (c) were additionally inhibited, wherever tested, by sugars lacking acetyl group. In the present study, HA-inhibition assays performed with PSA of D. a¤nis revealed that N-acetyl hexosamines (GlcNAc, (GlcNAc)2 , (GlcNAc)3 , GalNAc, ManNAc) which contain acetyl group on C-2 inhibited the agglutinating activity of PSA. On the other hand, their hexoses as well as hexosamine counterparts did not inhibit at all, thereby suggesting the

21

importance of acetyl group in agglutinin^ligand interaction. Moreover, inhibition of PSA by GlcNAc, (GlcNAc)2 and (GlcNAc)3 at an equimolar concentration clearly indicates that this agglutinin does not exhibit preference for anomeric con¢guration of GlcNAc, and it has a small combining site in contrast to other GlcNAc-speci¢c agglutinins [46^49]. The exquisite speci¢city of this agglutinin for acetyl group was further demonstrated when two sialic acids were examined as inhibitors, since NeuAc with acetyl group on C-5 was inhibitory, whereas NeuGc with the glycolyl group on C-5 was inactive. These observations unambiguously demonstrate that D. a¤nis agglutinin speci¢cally recognized the acetyl group in the carbohydrates independently of the presence of this group on C-2 (GlcNAc, (GlcNAc)2 , (GlcNAc)3 , GalNAc, ManNAc) or C-5 (NeuAc) and its stereochemical arrangement in the axial (ManNAc) or equatorial (GlcNAc, (GlcNAc)2 , (GlcNAc)3 , GalNAc, NeuAc) orientation. Interestingly, the speci¢city of this agglutinin to acetyl group is also consistent with the results obtained from inhibition tests with amino acids in which only acetylated amino acids containing N- or O-acetyl group inhibited the activity of PSA. Inhibition tests with glycoproteins revealed that BSM and fetuin were inhibitory, and these sialoglycoproteins are known to contain predominantly terminal NeuAc either K 2^6-linked to GalNAc (BSM) or K 2^3-linked to galactose (fetuin) in their oligosaccharide side chains [50,51]. The inhibitory potency of BSM and fetuin is presumably due to their terminal NeuAc, since porcine thyroglobulin and porcine stomach mucin, both contain terminal NeuGc [52,53], failed to inhibit the HA activity of PSA. In accordance with the inhibitory e¡ects observed in this study with free GalNAc and galactose, asialoBSM with terminal GalNAc continued to be inhibitory whereas asialo-fetuin terminated with galactose failed to inhibit the agglutinating activity. BSM has terminally both N- and O-acetyl sialic acids in the sugar chains, and de-O-acetylation of such sialoglycoproteins by base treatment speci¢cally hydrolyses O-acetyl groups of sialic acids without cleavage of peptide bonds [27,54]. In the present study, the inhibitory potency of BSM was reduced, but not abolished, after de-O-acetylation. Taken together, these ¢ndings suggest that D. a¤nis agglutinin recognizes

BBAGEN 24851 30-9-99

22

S. Murali et al. / Biochimica et Biophysica Acta 1472 (1999) 13^24

the terminal N- and O-acetyl groups in the oligosaccharide chain of glycoconjugates. PSA of D. a¤nis, as shown in other crustaceans [41,55^57], was also inhibited by LPS from diverse Gram-negative bacteria. The exact site in the bacterial LPS involved in the interaction with this agglutinin is not known at present. However, NeuAc, 2keto-3-deoxy-octonate (KDO, a molecule structurally similar with NeuAc) and other acetylated aminosugars do occur terminally in LPS coat of several strains of diverse bacterial species including Salmonella sp., E. coli, and Pseudomonas sp. [16,53,58,59]. It is, therefore, likely that the humoral agglutinin of D. a¤nis, with a distinct and unique speci¢city for Nand O-acetyl groups, could directly recognize and interact with invading and possibly pathogenic bacteria. This interaction might indicate a signi¢cant in vivo role of the D. a¤nis agglutinin in the host immune response against bacterial infections.

[5] [6]

[7]

[8]

[9] [10]

[11]

[12]

Acknowledgements S.M. is grateful to the Council of Scienti¢c and Industrial Research, New Delhi for the award of Senior Research Fellowship (No. 9/115 (329)/94EMR-I). We thank Dr. M.H. Ravindranath (John Wayne Institute for Cancer Treatment and Research, Santa Monica, CA) for constant encouragement and generous gift of several carbohydrates used in this study. We thank members of the Laboratory of Pathobiology for valuable help during the course of this study.

[13]

[14]

[15]

[16]

References

[17]

[1] R.W. Yeaton, Invertebrate lectins: I. Occurrence, Dev. Comp. Immunol. 5 (1981) 391^402. [2] N.A. Ratcli¡e, A.F. Rowley, S.W. Fitzgerald, C.P. Rhodes, Invertebrate immunity: basic concepts and recent advances, Int. Rev. Cytol. 97 (1985) 183^350. [3] L. Renwrantz, Lectins in molluscs and arthropods: their occurrence, origin and roles in immunity, in: A.M. Lackie (Ed.), Immune Mechanisms in Invertebrate Vectors, Clarendon Press, Oxford, 1986, pp. 81^93. [4] E.H. Richards, L.R. Renwrantz, Two lectins on the surface of Helix pomatia haemocytes: Ca2‡ -dependent, GalNac-speci¢c lectin and a Ca2‡ -independent, mannose 6-phosphate-

[18]

[19] [20]

[21]

speci¢c lectin which recognises activated homologous opsonins, J. Comp. Physiol. 161B (1991) 43^54. V.J. Smith, R.S. Chisholm, Non-cellular immunity in crustaceans, Fish Shell¢sh Immunol. 2 (1992) 1^31. P. Mullainadhan, L. Renwrantz, Lectin-dependent recognition of foreign cells by hemocytes of the mussel, Mytilus edulis, Immunobiology 171 (1986) 263^273. T. Suzuki, K. Mori, Immunolocalization and in vitro secretion of hemolymph lectin of the pearl oyster, Pinctada fucata martensii, Zool. Sci. 8 (1991) 23^29. D.W. Fountain, B.A. Campbell, A lectin isolated from mucus of Helix aspersa, Comp. Biochem. Physiol. 77B (1984) 419^429. D.R. Coombe, P.L. Ey, C.R. Jenkin, Self/non-self recognition in invertebrates, Quart. Rev. Biol. 59 (1984) 231^255. J.A. Olafsen, Role of lectins in invertebrate humoral defense, in: W.S. Fisher (Ed.), Disease Processes in Marine Bivalve Molluscs, Am. Fish. Soc. Special Publ. 18, 1988, pp. 189^ 205. A.M. Lackie, G.R. Vasta, The role of galactosyl-binding lectin in the cellular immune response of the cockroach Periplaneta americana (Dictyoptera), Immunology 64 (1988) 353^357. G.R. Vasta, The multiple biological roles of invertebrate lectins: their participation in nonself recognition mechanisms, in: G.R. Warr, N. Cohen (Eds.), Phylogenesis of Immune Functions, CRC Press, Boca Raton, FL, 1991, pp. 73^101. P.D.Sr. Mercy, M.H. Ravindranath, Hemolysis and clearance of erythrocytes in Scylla serrata are related to the agglutination by the native sialic acid-speci¢c lectin, Comp. Biochem. Physiol. 109A (1994) 1075^1083. G. Sen, M. Chowdhury, C. Mandal, O-Acetylated sialic acid as a distinct marker for di¡erentiation between several leukemia erythrocytes, Mol. Cell. Biochem. 136 (1994) 65^ 70. M.H. Ravindranath, J.C. Paulson, R.F. Irie, Human melanoma antigen O-acetylated ganglioside GD3 is recognized by Cancer antennarius lectin, J. Biol. Chem. 263 (1988) 2079^ 2086. M. Slifkin, R.J. Doyle, Lectins and their application to clinical microbiology, Clin. Microbiol. Rev. 3 (1990) 197^218. R. Uchikawa, E.S. Loker, Lectin-binding properties of the surfaces of in vitro transformed Schistosoma mansoni and Echinostoma paraensei sporocysts, J. Parasitol. 77 (1991) 742^748. B.S. Mitchell, U. Schumacher, The use of lectin Helix pomatia agglutinin (HPA) as a prognostic indicator and as a tool in cancer research, Histol. Histopathol. 14 (1999) 217^226. N. Sharon, H. Lis, Lectins as cell recognition molecules, Science 46 (1989) 227^234. H. Bretting, E.A. Kabat, Puri¢cation and characterization of agglutinins from the sponge Axinella polypoides and a study of their combining sites, Biochemistry 15 (1976) 3228^3236. M.H. Ravindranath, H.H. Higa, E.L. Cooper, J.C. Paulson, Puri¢cation and characterization of an O-acetylsialic acid-

BBAGEN 24851 30-9-99

S. Murali et al. / Biochimica et Biophysica Acta 1472 (1999) 13^24

[22]

[23]

[24]

[25]

[26]

[27] [28] [29]

[30]

[31]

[32]

[33]

[34]

[35] [36]

[37]

[38]

[39]

[40]

speci¢c lectin from a marine crab Cancer antennarius, J. Biol. Chem. 260 (1985) 8850^8856. B. Mauchamp, Puri¢cation of an N-acetyl-D-glucosamine speci¢c lectin (P.B.A.) from epidermal cell membranes of Pieris brassicae L, Biochimie 64 (1982) 1001^1008. O.M. Koch, C.K. Lee, G. Uhlenbruck, Cerianthin lectins: a new group of agglutinins from Cerianthus membranaceus, Immunobiology 163 (1982) 53^62. M. Faglioni, V. Parisi, G. Salsi, Some experimental observations on the heteroagglutinins of decapod crustaceans, Boll. Zool. 38 (1971) 159^163. G.R. Vasta, J.J. Marchalonis, Humoral recognition factors in the Arthropoda. The speci¢city of Chelicerata serum lectins, Am. Zool. 23 (1983) 157^171. S. Murali, P. Mullainadhan, M. Arumugam, A lipopolysaccharide-binding hemagglutinin with speci¢city for acetylated aminosugars in the serum of the hermit crab Diogenes a¤nis (Henderson), J. Invertebr. Pathol. 64 (1994) 221^227. A.H. Sarris, G.E. Palade, The sialoglycoproteins of murine erythrocyte ghosts, J. Biol. Chem. 254 (1979) 6724^6731. J.S. Garvey, N.E. Cremer, D.H. Sussdorf, Methods in Immunology, W.A. Benjamin, Reading, MA, 1979. P. Vretblad, Puri¢cation of lectins by biospeci¢c a¤nity chromatography, Biochim. Biophys. Acta 434 (1976) 169^ 176. L.A. Osterman, Methods of Protein and Nucleic acid Research, Vol. 3, Chromatography, Springer, Berlin, 1986, pp. 308^404. O.H. Lowry, N. Rosenbrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin-phenol reagent, J. Biol. Chem. 193 (1951) 265^275. H.R. Maurer, Disc Electrophoresis and Related Techniques of Polyacrylamide Gel Electrophoresis, Walter de Gruyter, Berlin, 1971. W. Ansorge, Fast and sensitive detection of protein and DNA bands by treatment with potassium permanganate, J. Biochem. Biophys. Methods 11 (1985) 13^20. U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4 , Nature 227 (1970) 680^685. O. Ouchterlony, Di¡usion in gel methods for immunological analysis, Prog. Allergy 6 (1962) 30^154. T. Suzuki, S. Natori, Identi¢cation of a protein having hemagglutinating activity in the hemolymph of the silkworm, Bombyx mori, J. Biochem. 93 (1983) 583^590. M. Schwyzer, R.L. Hill, Porcine A blood group speci¢c N-acetylgalactosaminyltransferase : I. Puri¢cation from porcine submaxillary glands, J. Biol. Chem. 252 (1977) 2338^ 2345. K. Drickamer, Ca2‡ -dependent carbohydrate-recognition domains in animal proteins, Curr. Opin. Struct. Biol. 3 (1993) 393^400. J.L. Hall, D.T. Rowlands, Heterogeneity of lobster agglutinins. II. Speci¢city of agglutinin-erythrocyte binding, Biochemistry 13 (1974) 828^832. S. Ratanapo, M. Chulavatnatol, Monodin, a new sialic acid-

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48]

[49]

[50] [51]

[52]

[53] [54]

[55]

[56]

[57]

23

speci¢c lectin from black tiger prawn (Penaeus monodon), Comp. Biochem. Physiol. 97B (1990) 515^520. F. Vargas-Albores, M.A. Guzma¨n, J.L. Ochoa, A lipopolysaccharide-binding agglutinin isolated from brown shrimp (Penaeus californiensis Holmes) haemolymph, Comp. Biochem. Physiol. 104B (1993) 407^413. G. Fragkiadakis, E. Stratakis, Characterization of hemolymph lectins in the prawn Parapenaeus longirostris, J. Invertebr. Pathol. 65 (1995) 111^117. K. Muramoto, T. Matsuda, K. Nakada, H. Kamiya, Occurrence of multiple lectins in the hemolymph of kuruma prawn Penaeus japonicus, Fish. Sci. 61 (1995) 131^135. K. Umetsu, S. Kosaka, S. Kashimura, T. Suzuki, Studies on anti-B speci¢city and heterogeneity of some invertebrate hemagglutinins, Tohoku J. Exp. Med. 129 (1979) 161^167. F.J. Cassels, J.J. Marchalonis, G.R. Vasta, Heterogeneous humoral and hemocyte-associated lectins with N-acylaminosugar speci¢cities from the blue crab, Callinectes sapidus Rathbun, Comp. Biochem. Physiol. 85B (1986) 23^30. A.K. Allen, A. Neuberger, The puri¢cation and properties of the lectin from potato tubers, a hydroxy-containing glycoprotein, Biochem. J. 135 (1973) 307^314. I.J. Goldstein, S.T. Hammarstro«m, G. Sundblad, Precipitation and carbohydrate-binding speci¢city studies on wheat germ agglutinin, Biochim. Biophys. Acta 405 (1975) 53^ 61. H. Bretting, E.A. Kabat, J. Liao, M.E.A. Pereira, Puri¢cation and characterization of the agglutinins from the sponge Aaptos papillata and a study of their combining sites, Biochemistry 15 (1976) 5029^5038. A. Mo«ck, L. Renwrantz, Isolation and characterization of a lectin from the cephalochordate Branchiostoma lanceolatum (Pallas), Comp. Biochem. Physiol. 99B (1991) 699^701. T. Tsuji, T. Osawa, Carbohydrate structures of bovine submaxillary mucin, Carbohydr. Res. 151 (1986) 391^402. B. Nilsson, N.E. Norde¨n, S. Svensson, Structural studies on the carbohydrate portion of fetuin, J. Biol. Chem. 254 (1979) 4545^4553. M.T. McQuillan, V.M. Trikojus, Thyroglobulins, in: A. Gottschalk (Ed.), Glycoproteins, Elsevier, Amsterdam, 1966, pp. 463^515. R. Schauer, Sialic Acids: Chemistry, Metabolism and Function, Springer, Wien, 1982. R. Schauer, Chemistry, metabolism and biological functions of sialic acids, Adv. Carbohydr. Chem. Biochem. 40 (1982) 131^234. G.A. Fragkiadakis, E.M. Stratakis, The lectin from the crustacean Liocarcinus depurator recognizes O-acetylsialic acids, Comp. Biochem. Physiol. 117B (1997) 545^552. P. Kopa¨cªek, L. Grubho¡er, K. So«derha«ll, Isolation and characterization of a hemagglutinin with a¤nity for lipopolysaccharides from plasma of the cray¢sh Pacifastacus leniusculus, Dev. Comp. Immunol. 17 (1993) 407^418. R. Maheswari, P. Mullainadhan, M. Arumugam, Characterisation of a natural haemagglutinin with a¤nity for acetylated aminosugars in the serum of the marine prawn, Penaeus

BBAGEN 24851 30-9-99

24

S. Murali et al. / Biochimica et Biophysica Acta 1472 (1999) 13^24

indicus (H. Milne Edwards), Fish Shell¢sh Immunol. 7 (1997) 17^28. [58] O. Lu«deritz, J. Gmeiner, B. Kickhofen, H. Mayer, O. Westphal, R.W. Wheat, Identi¢cation of D-mannosamine and quinovosamine in Salmonella and related bacteria, J. Bacteriol. 95 (1968) 490^494.

[59] A. Gamian, L. Kenne, M. Mieszala, J. Ulrich, J. Defaye, Structure of the Escherichia coli O24 and O56 O-speci¢c sialic-acid-containing polysaccharides and linkage of these structures to the core region in lipopolysaccharides, Eur. J. Biochem. 225 (1994) 1211^1220.

BBAGEN 24851 30-9-99