Characterisation of monoclonal antibodies to separate epitopes on salmon IgM heavy chain

Fish & Shellfish Immunology (1996) 6, 185–198

Characterisation of monoclonal antibodies to separate epitopes on salmon IgM heavy chain BERGLJOu T MAGNADOu TTIR, HELGA KRISTJAu NSDOu TTIR* SIGRIÐUR GUÐMUNDSDOu TTIR

AND

Institute for Experimental Pathology, University of Iceland, Keldur v./Vesturlandsveg, IS-112 Reykjavı´k, Iceland, and *Department of Immunology, University Hospital, IS-101 Reykjavı´k, Iceland (Received 1 August 1995, accepted in revised form 6 November 1995) Two types of monoclonal antibodies (MAbs) to salmon IgM heavy chain, MAb type I and II, were characterised with respect to their reaction with di#erent enzyme fragments of IgM. Protocols were devised for trypsin, pepsin and papain digestion of salmon IgM. Trypsin and pepsin digestion yielded mainly 25, 30·5 kDa and 52 kDa breakdown fragments of the heavy chain after reduction and denaturation. Papain gave a predominant 15·2 kDa fragment and minor 52 and 30·5 kDa fragments. Amino acid sequence analysis of the 30·5 kDa heavy chain fragment identified it as domains Cì3 and Cì4 of the Fc tail of salmon IgM. MAb type I reacted with the 30·5 and 52 kDa fragments of the heavy chain indicating specificity for the Fc region. From this it was concluded that these two fragments, sharing a common epitope, both belonged to the Fc tail. MAb type II reacted with the 25 kDa fragment of the heavy chain. It was concluded that this fragment was the Fab (Fd) region of the ì chain. The flow cytometry analysis supports the evidence for the separate epitope specificity of these two monoclonal antibodies. MAb type II, the anti-Fab antibody, selected IgM bearing leucocytes more e$ciently than the anti-Fc antibody, MAb type I. ? 1996 Academic Press Limited Key words:

Atlantic salmon, IgM, Fab, Fc, trypsin, pepsin, papain, monoclonal antibodies, flow cytometry, magnetic cell sorting.

I. Introduction Specific antibodies, polyclonal and/or monoclonal, to immunoglobulins and their sub units have proved to be valuable tools in immunological research and in immunological assays. In the last decade these have also been extensively used in various studies of the immune system of fish (DeLuca et al., 1983; Sanchez, et al., 1989; Thuvander et al., 1990; Israelsson et al., 1991; Magnadottir & Gudmundsdottir, 1992; Estevez et al., 1994). Since the pioneering work by Porter (1959), enzymatic cleavage has been the basic research tool in studies of the structure and function of immunoglobulins. The structure and function of IgM is of special interest, this being the first immunoglobulin to appear in evolution and the only immunoglobulin class of lower vertebrates. It has been demonstrated that there is a significant 185 1050–4648/96/030185+14 $18.00/0

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homology between the amino acid sequence of IgM ì chain from very diverse species (Rosenhein et al., 1985; Bengtén et al., 1991; Andersson & Matsunaga, 1993; Lee et al., 1993). However, there is su$cient variation to result in, for example, variable sensitivity to enzymatic digestion (pers. observation). A single protocol for enzymatic digestion of IgM from all species is therefore not likely to be devised but has to be arrived at experimentally in each case (Klapper et al., 1971; Beale & van Dort, 1982; Beale, 1987; Coosemans et al., 1989; van Ginkel et al., 1991; Glynn & Pulsford, 1993). The aim of this paper is to characterise the epitope specificity of monoclonal antibodies produced against salmon IgM and to determine their usefulness in leucocyte sorting. Devising protocols for the proteolytic digestion of salmon IgM was an important element in this study.

II. Materials and Methods PREPARATION OF SALMON IgM

Salmon IgM was isolated from pooled serum, collected from healthy Atlantic salmon (Salmo salar L.) and stored at "50)C. The method of purification has been described elsewhere (Magnadottir, 1990). Briefly, untreated serum was passed through a CM A$ Gel Blue column (BioRad) and the unbound material was collected. This was followed by ammonium sulphate precipitation and gel filtration of the dissolved and dialysed precipitate on a Superose 6 column (HR10/30, Pharmacia) using the FPLC system from Pharmacia. Purity was checked by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and by immunoelectrophoresis with rabbit antibodies to salmon serum and polyclonal mouse antibodies to salmon IgM (Hudson & Hay, 1989).

PROTEIN MEASUREMENT

The concentration of purified immunoglobulin was calculated from the optical density of the solution at 280 nm using an extinction coe$cient of E1 cm =13·7 for 1% solution of salmon IgM (Williams & Chase, 1968). PRODUCTION OF POLYCLONAL ANTIBODIES TO SALMON IgM (PAb)

Polyclonal antibody (PAb) to salmon IgM was prepared in mouse ascitic fluid using the method described by Overkamp et al. (1988). The ascitic fluid was clarified by centrifugation at 750 g, filtered through a 0·45 ìm filter (Millipore corp.) and stored at "70)C. Working solutions were mixed with an equal volume of glycerol and stored at "20)C. The anti-IgM activity was measured using standard ELISA procedure on IgM coated trays (1 ìg well "1). Antibody titre was defined as the reciprocal value of the dilution that gave optical density readings of about 2–3#the bu#er control at 492 nm. The specificity was checked by immunoelectrophoresis with purified salmon IgM and salmon serum (Hudson & Hay, 1989).

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PRODUCTION OF MONOCLONAL ANTI SALMON IgM ANTIBODIES

Standard procedure was used for the monoclonal antibody (MAb) production (Campbell, 1986) and over 50 hybridoma, yielding relatively high levels of anti-salmon IgM activity, were collected and stored in liquid nitrogen. Anti-IgM activity was measured using standard ELISA procedure. The 10–15 most active hybridoma were cloned and the antibody production of those that maintained a high level of anti-IgM activity after having been cloned three times, was amplified in mouse ascitic fluid. The ascitic fluid was clarified, filtered, titrated and stored as described above. After preliminary examination by Western blotting two MAbs, MAb-type I (clone 1206) and MAb-type II (clone 202), were selected for further characterisation. DETERMINATION OF IMMUNOGLOBULIN CLASS

To determine the mouse antibody immunoglobulin class, a MAb isotyping kit from Boehringer Mannheim was used. The class determination was carried out on hybridoma culture fluid. PROTEOLYTIC FRAGMENTATION

Enzymes, coupled to actigel-ald from Sterogene Biochemicals, were used for the proteolytic digestion of IgM. The manufacturer’s instructions formed the basis of protocols devised after extensive trials. These involved varying the IgM: enzyme-gel ratio, changing the pH, using di#erent incubation temperature and duration, and in the case of papain the presence of, or activation by thiols was tested (McIlroy & Stevenson, 1974). The protocols described below were aimed at obtaining stable intermediate breakdown fragments in su$cient concentration to allow detection by silver staining and by Western blotting using the antibodies under study. Trypsin digestion Trypsin-actigel, which contained approximately 1 mg enzyme ml "1 gel (1 ml was equivalent to approximately 570 mg of suction dry gel), was equilibrated with several bed volumes of 0·1 M Tris-HCl bu#er, pH 8·0, containing 10 mM CaCl2 on a glass filter and finally suction dried. Pure salmon IgM, concentration 1 mg ml "1, was equilibrated in the same bu#er in a prodialyser (ISS-enprotech) for 1 h. Fifty mg of suction dry gel, equivalent to approximately 85 ìg of trypsin, were mixed with 75 ìl of the equilibrating bu#er and 50 ìl (50 ìg) of IgM solution and incubated overnight (§16 h) at 45)C with occasional shaking. A control omitting the trypsin-gel was included. After the incubation period the sample was centrifuged at 200 g for 10 min and the supernatant collected. Pepsin digestion Pepsin-actigel, which contained approximately 3–4 mg enzyme ml "1 gel, was equilibrated with several bed volumes of 20 mM sodium acetate bu#er, pH 5·5–6·0, on a glass filter and suction dried. Pure salmon IgM, concentration

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1 mg ml "1, was equilibrated in the same bu#er in a prodialyser for 1 h. Ten mg of suction dry gel, equivalent to approximately 50–70 ìg of pepsin, were mixed with 75 ìl of the equilibrating bu#er and 50 ìl (50 ìg) of IgM solution and incubated at 37)C for 1 h with occasional shaking. After the incubation the sample was centrifuged as above and the supernatant collected. A control omitting the pepsin-gel was included. Papain digestion Papain-actigel, which contained approximately 2 mg enzyme ml "1 gel, was equilibrated in 20 mM phosphate bu#er, pH 6·2 containing 1 mM dithiothreitol and 5 mM EDTA, on a glass filter. Pure salmon IgM, at a concentration of 1 mg ml "1, was equilibrated in the same bu#er in a prodialyser for 1 h. Ten mg of suction dry gel, equivalent to approximately 34 ìg enzyme, were mixed with 75 ìl of the equilibrating bu#er and 50 ìl (50 ìg) of IgM. The sample was incubated at 37)C for 1 h with occasional shaking. After the incubation the sample was centrifuged as above and the supernatant collected. A control omitting the papain-gel was included. SDS-PAGE AND WESTERN BLOTTING

Before SDS-PAGE, samples were reduced and denatured in 0·5% 2-mercaptoethanol and 2% SDS for 2–5 min at 100)C. A Mini-PROTEAN II system was used (Bio Rad Laboratories) and, following the manufacturer’s instructions, electrophoresis was carried out in 25 mM Tris-glycine bu#er, pH 8·8, containing 0·1% SDS. The stacking gel was 4·5% and the resolving gel 14% acrylamide. Molecular weights of the IgM fragments were estimated, after SDS PAGE separation and silver staining (silver staining kit from Bio Rad), by plotting a graph of log molecular weight of standard proteins (Sigma) against their Rf values and interpolating the unknown values from this graph. After electrophoresis, transfer onto nitrocellulose paper (Hybond-ECL, Amersham) was carried out using the Mini Trans-Blot module from Bio Rad Laboratories, according to the manufacturer’s instructions. Transfer was for 90 min at 4)C in 25 mM Tris-glycine bu#er, pH 8·8, containing 20% methanol. The blots were immunostained using an enhanced chemiluminescence (ECL) Western blotting detection system from Amersham. The residual sites of the blot were blocked with Tris bu#ered saline (TBS), pH 7·8, containing 2% bovine serum albumin (BSA), 2% normal goat serum and 0·1% Tween, the primary antibody being polyclonal or monoclonal anti IgM antibody, diluted 1/2000, and the secondary antibody being horseradish peroxidase conjugated sheep anti mouse antibody from Amersham, diluted 1/1000. Prestained standards from Bio Rad were used in Western blotting. CARBOHYDRATE REMOVAL BY N-GLYCOSIDASE

The enzyme PNGase F, from BioLabs, was used to remove N-linked oligosaccharide from reduced salmon IgM. The reaction conditions used were as suggested by the enzyme manufacturers. 50–100 Units of PNGase were used to

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digest 1 ìg IgM. After the carbohydrate removal SDS-PAGE and Western blotting was carried out as described above and the e#ect on the binding specificity of the MAb examined. AMINO ACID SEQUENCE ANALYSIS OF THE 30·5 kDa TRYPSIN FRAGMENT OF IgM HEAVY CHAIN

Salmon IgM (750 ìg) was treated with trypsin as described above, the resulting fragments were separated by preparative SDS-PAGE and transferred to Immobilon-PSQ membrane (Millipor) by Western blotting. The blot was then developed with 0·1% solution of Amido black in 40% methanol and 1% acetic acid for 1 min. Distilled water was used for destaining and the 30·5 kDa trypsin fragment cut from the blot. The amino acid sequence analysis of this fragment was carried out using an Applied Biosystems 473A automated protein sequencer. This work was done by Mr. Kevin Bailey, Biopolymer Synthesis and Analysis Unit, Department of Biochemistry, University of Nottingham, England. ISOLATION OF LEUCOCYTES FROM BLOOD

Atlantic salmon was anaesthetised with benzocaine (40 mg 1 "1) and blood sampled from the caudal aorta. For flow cytometry, samples from four fish weighing on average 60 g were pooled, but for magnetic cell sorting blood was collected from fish weighing 500 g. Blood samples were diluted 31 in Hanks’ balanced salt solution (HBSS) containing 50 AE heparin ml "1, 100 iu ml "1 penicillin and 50 iu ml "1 streptomycin. One part of the diluted blood sample was layered onto two parts of 54% Percoll (Pharamacia) in 0·9% NaCl and centrifuged at 500 g for 20 min at room temperature. The leucocyte fraction, at the interface between the gradient and the plasma, was harvested and washed twice in HBSS. The cells were resuspended in Leibowitz-15 medium (Gibco) with 5% foetal calf serum (FCS, Myoclone, Gibco), 2 mM glutamine and penicillin and streptomycin as described above. FLOW CYTOMETRY

Leucocytes (750 000) from salmon blood samples were centrifuged at 300 g for 10 min at 4)C and resuspended in 50 ìl of 0·01 M phosphate bu#ered saline, pH 7·4 (PBS). For staining 50 ìl of MAb or PAb mouse antibodies against salmon IgM (ascites), diluted in PBS, was added to the cells and incubated on ice for 30 min. After two washes with PBS containing 0·5% BSA and 0·1% sodium azide (PBS-BSA), the cells were stained with a fluorescent second antibody. Fifty ìl of FITC-conjugated rabbit anti-mouse F(ab’)2 (Dakopat) were added to the cell suspension and incubated on ice for 30 min. After one wash the cells were resuspended in 500 ìl of the washing bu#er with 0·5% formaldehyde. The samples were subsequently collected on a flow cytometer (FACScan, Becton and Dickinson). For each sample 10 000 cells were collected. The data was then first analysed on a FSC/SSC dot plot and lymphocytes were gated for further analyses of fluorescence. Histograms for FL1 (FITC) were used to analyse and compare the fluorescent staining of the cells for the two types of MAb and the negative control.

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MAGNETIC CELL SORTING (MACS)

The Milteny Biotec GmbH system was used and separation was according to the manufacturers instructions. Blood leucocytes, 107 cells, were incubated with MAb against salmon IgM in PBS-BSA bu#er at 2–8)C for 15 min, the final volume being 80 ìl, and then washed once in the same bu#er. The cells were resuspended in 80 ìl of PBS-BSA and 20 ìl of MACS microbeads (goat anti-mouse Ig) added and incubated at 2–8)C for 15 min. After one wash, the cells were layered onto a steel wool column and placed in a magnetic field. Unbound, surface Ig negative (Ig " ) cells were washed through the column with 2–4 volumes of PBS-BSA through a 24G needle. Loosely bound cells were flushed up through the column (outside the magnetic field) placed in the magnetic field again and washed o# the column using a larger diameter needle (22G). Finally the column was removed from the magnetic field and surface Ig positive (Ig + ) cells flushed out with PBS-BSA. The separated cell populations were washed in HBSS and counted.

III. Results ENZYMATIC CLEAVAGE OF SALMON IgM

Untreated IgM split into its 72 kDa heavy chain and 26·5 kDa light chain components after reduction, as demonstrated by silver staining (Fig. 1, lane 2) and Western blotting with PAb anti-IgM antibody (Fig. 2, lane 2). Most of the PAbs produced showed stronger a$nity for the heavy chain than for the light chain and the intensity of reaction with various breakdown fragments described below varied slightly. After trypsin digestion there was some reduction of the 72 kDa heavy chain band, accompanied by the appearance of several breakdown fragments. The main band, as indicated by silver staining, was 30·5 kDa and an apparent increase in the density and widening of the light chain band (25 kDa) was also seen (Fig. 1, lane 3). In addition PAb detected a major 52 kDa fragment and several other fainter fragments (Fig. 2, lane 3). After pepsin digestion the major fragment was 52 kDa and increased density of the 25 kDa band was also observed after silver staining (Fig. 1, lane 4). PAb gave a similar picture (Fig. 2, lane 4) but some PAb also detected a 30·5 kDa fragment (results not shown). After papain digestion there was a considerable reduction of the heavy chain and the light chain was not detectable. The main breakdown fragment seen after silver staining was 15·2 kDa (Fig. 1, lane 5). This fragment was not detected by the PAb after Western blotting. PAb did, however, detect a relatively strong 52 kDa fragment and a fainter 30·5 kDa band (Fig. 2, lane 5). CHARACTERISATION OF THE 30·5 kDa TRYPSIN FRAGMENT OF IgM HEAVY CHAIN

Ten residues were analysed from the N-terminal region and two sequences were identified: T V G Y T S S D A G and R T V G Y T S S D A. These sequences correspond to residues 206–215 and 205–214 of the constant region of salmon

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Fig. 1. SDS PAGE analysis (silver staining) of control and enzyme digested salmon IgM, after reduction. Lane 1: Molecular weight markers (kDa). Lane 2: Control IgM. Lane 3: Trypsin digested IgM. Lane 4: Pepsin digested IgM. Lane 5: Papain digested IgM.

IgM ì chain, from the junction of domain Cì2 and Cì3. This is according to the amino acid sequence analysis by Hordvik et al. (1992). The two sequences result from incomplete digestion in this area due to two arginine (R) residues available for trypsin cleavage just on the left of the first sequence.

CHARACTERISATION OF THE MONOCLONAL ANTIBODIES

MAb-type I and II were both heavy chain specific (Figs 3 & 4, lane 2). MAb-type I reacted with the 30·5 and 52 kDa fragments obtained after trypsin, pepsin and papain digestion (Fig. 3, lanes 3–5) whereas MAb-type II reacted with the 24·5–25 kDa breakdown fragment seen after trypsin and pepsin digestion (Fig. 4, lanes 3 and 4). Neither the MAb-type I nor type II reacted with the 15·2 kDa papain fragment seen in Figure 1, lane 5. Removal of N-linked oligosaccharide from the salmon IgM heavy chain, approximately 10 kDa, did not a#ect the binding specificity of the MAbs in Western blotting (results not shown). MAb-type I was Ig subclass IgG2b and MAb-type II was subclass IgG1. The titre values of both MAb types and of PAb ascitic fluids were 3–6#105.

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Fig. 2. Western blotting of control and enzyme digested salmon IgM, after reduction. Immunostained with polyclonal antibody (PAb) to IgM. Lane 1: Molecular weight markers (kDa). Lane 2: Control IgM. Lane 3: Trypsin digested IgM. Lane 4: Pepsin digested IgM. Lane 5: Papain digested IgM. FLOW CYTOMETRY AND CELL SORTING

The results of the flowcytometric analysis showed that 33% of the defined lymphocytes were positive when stained with MAb type II [Fig. 5(b)] and 0·11% when stained with MAb-type I [Fig. 5(c)] or similar to the negative control [0·14%, Fig. 5(d)]. The MAb-type II was further tested in two separate magnetic cell sorting experiments, as shown in Table 1. In the first experiment, 75·3% of the original number of leucocytes were recovered while 96·3% were recovered in the second experiment. The percentage of Ig + cells in the second experiment was 37·0% which corresponded to the results obtained with flow cytometry (Fig. 5). MAb-type II selected Ig + cells as e#ectively as PAb (results not shown). IV. Discussion The main fragments of the salmon IgM heavy chain obtained after proteolytic digestion were 52, 30·5, 25 and 15·2 kDa. The 52 and 30·5 kDa fragments had a common epitope, which MAb-type I reacted with, and the 25 kDa piece

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Fig. 3. Western blotting of control and enzyme digested salmon IgM, after reduction. Immunostained with MAb-type I. Lane 1: Molecular weight markers (kDa). Lane 2: Control IgM. Lane 3: Trypsin digested IgM. Lane 4: Pepsin digested IgM. Lane 5: Papain digested IgM.

had a separate epitope, for which MAb-type II was specific. The 15·2 kDa papain fragment was not detected by either the MAb or the PAb in Western blotting. Amino acid sequence analysis identified the 30·5 kDa fragment as being the last two domains of the Fc tail of salmon IgM (domain Cì3 and Cì4) which means that the MAb-type I was specific for an epitope on the Fc region. The separate epitope specificity of MAb-type II and the size of the remaining heavy chain fragment, 25 kDa, indicates that this fragment is the Fab (Fd) region of the heavy chain. It was therefore concluded that MAb-type II was specific for an epitope on the Fab region. The results of the flow cytometry and cell sorting supports the evidence for the separate epitope specificity of these two MAb. MAb-type II, the anti Fab antibody, selected IgM bearing leucocytes whereas MAb-type I, the anti Fc antibody, did not. Since domain Cì4 is missing on the membrane bound IgM in teleosts (Bengtén et al., 1991; Hordvik et al., 1992; Anderson & Matsunaga, 1993; Lee et al., 1993) the reason for the poor selection by MAb-type I could be an indication that its specific epitope lies on the Cì4 domain of the secreted Ig (serum IgM). Steric hindrance could also be involved, the Fc region on the membrane bound IgM being less accessible to the antibody than the Fab region. The Fc and Fab regions of IgM have important separate biological functions. The Fc region mediates binding of the Ig to host tissues, including

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Fig. 4. Western blotting of control and enzyme digested salmon IgM, after reduction. Immunostained with MAb-type II. Lane 1: Molecular weight markers (kDa). Lane 2: Control IgM. Lane 3: Trypsin digested IgM. Lane 4: Pepsin digested IgM. Lane 5: Papain digested IgM.

various cells of the immune system and components of the complement system. The antigen binding site on the other hand is associated with the variable domains of the Fab region (Day, 1990). MAb to these regions are therefore valuable tools in further studies of the immune system of salmon. Salmon IgM was relatively resistant to trypsin digestion and it was found that the selected temperature of 45)C was a key factor for successful, though partial, digestion. The importance of high temperature for successful tryptic cleavage of IgM was first described by Plaut and Tomasi (1970). The high temperature is believed to expose the Cì1–Cì2 junction to enzymatic digestion and at the same time stabilise all or a proportion of the Fc tail (Plaut & Tomasi, 1970; Siegel et al., 1981; Haynes et al., 1988; Day, 1990). When salmon IgM was exposed to trypsin at 45)C the prime site of cleavage could have been the two lysine residues near the junction of Cì1 and Cì2. The exposed Cì2 domain, under these conditions, then seemed sensitive to further trypsin digestion, the main yield being the relatively stable Cì3 and Cì4 end (30·5 kDa) of the Fc tail.

EPITOPE SPECIFICITY OF MAbs

MabII (202)

R1: lymphocyte gate SSC\side scatter

250

195

100

(a)

(b)

200 150 50

100 50 50 100 150 200 FSC\forward scatter

0

250

0 100

MabI (1206)

102

103

104

103

104

Negative control

100

100 (c)

50

0 100

101

(d)

50

101

102

103

104

0 100

101

102

Fig. 5. Staining of lymphoid cells with MAb and flowcytometric analysis. (a) FSC/SSC distribution of salmon blood cells with a gate defining the lymphocyte population. (b)–(d) show the distribution of fluorescence (FL1) of lymphocytes after staining with FITC-conjugated MAb and a negative control for the staining procedure. (b) Stained with MAb-type II, (c) stained with MAb-type 1, and (d) non-stained lymphocytes.

Similar results were obtained by van Ginkel et al. (1991) when subjecting Channel catfish IgM to tryptic digestion at 37)C and by Glynn and Pulsford (1993) treating flounder IgM with trypsin at 56)C. Klapper et al. (1971), on the other hand, found that the Fc tail of lemon shark IgM was completely degraded by trypsin at 37)C. With respect to the enzyme:IgM ratio, the incubation time and optimum temperature used, salmon IgM appeared to be more sensitive to pepsin than to trypsin digestion. This was the case even though it was found necessary to raise the pH of the equilibrating and incubating bu#er from the optimum pH 4·0–4·5 to the nearly inhibitory pH of 5·5–6·0, to avoid irreversible precipitation of salmon IgM. As in the case of the trypsin digestion, the prime site of cleavage was the junction of domains Cì1 and Cì2. However, since higher yields of the 52 kDa fragment were obtained than of the 30·5 kDa piece after pepsin digestion it is concluded that the CH2 domain was relatively more resistant to further degradation by pepsin than by trypsin. Papain cleavage of salmon IgM took place in the presence of a reducing agent (dithiothreitol). When incubated completely free of the reducing agent

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Table 1. Leucocytes, isolated from Atlantic salmon blood samples, stained with MAb-type II and separated by MACS into surface Ig-negative (Ig " ) and surface Ig-positive (Ig + ) sub-populations Total number of cells#106

Experiment 1 Experiment 2

Starting number

Ig "

washed o#

Ig +

30·0 27·0

15·0 15·2

2·4 0·8

5·2 10·0

or with dithiothreitol-sensitised papain according to the method by McIlroy and Stevenson (1974) no enzymatic digestion occurred. This suggests that the salmon IgM molecule had to be at least partially split into its heavy and light chain components for papain susceptible sites to be exposed. Once exposed, however, both the light chain and the Fab part of the heavy chain (Fd) were particularly susceptible to papain digestion whereas the Fc tail was slightly more resistant. The 15·2 kDa papain breakdown fragment was not identified. It has been shown that salmon IgM is heterogeneous with respect to overall charge (Håvarstein et al., 1988) and Hordvik et al. (1992) have since demonstrated that this heterogeneity has its genetic origin in two isotypic IgM heavy chain genes. It was not attempted in this study to carry out the protease digestion on the separate isotypes. It is possible that the two isotypic IgM have di#erent sensitivity to these proteases and which might account for the partial digestion observed. Trypsin, pepsin and papain are enzymes traditionally used when studying the structure of Ig (Day, 1990). Their action is influenced by their substrate specificity and optimum pH requirement (Carrey, 1989). The specificity of these enzymes is, for example, for arginine (R) and lysine (K) (trypsin and papain), phenylalanine (F) (pepsin and papain) and leucine (L) (pepsin). When the amino acid sequence of salmon constant ì region is examined for these amino acids it is clear that a multitude of possible cleavage points are available (Hordvik et al., 1992). However, other factors such as the basic folding pattern of the protein and degree and nature of glycosylation also play a role, making the enzyme fragmentation less predictable (Beale & van Dort, 1982; Beale, 1987). It is hoped that the methods devised here for the production of Fab and Fc fragments of salmon IgM by proteolytic digestion will prove valuable in further studies of the structure and function of salmon IgM. They will also be a useful basis of protocols for similar proteolytic digestion of other IgM species. The authors wish to thank R. Lutley, formerly at the Institute for Experimental Pathology, Keldur for initial participation in this project and E. Gunnarsson, Institute for Experimental Pathology, Keldur, for preparing polyclonal and monoclonal ascites. Thanks are also due to L. Hammarström, Huddinge University Hospital, Sweden, for an analysis of the proteolytic sites and to K. Bailey, University of Nottingham, for valuable comments on the amino acid sequence analysis. This work was supported by a grant from the Icelandic Council of Science no. 90L02.

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