investigation of the heterogeneity of rhesus monkey alpha1-antitrypsin

Comp. Biochem. PhysioL Vol. 85B, No. 2, pp. 325-332, 1986 0305-0491/86 $3.00 + 0.00 Printed in Great Britain Pergamon Journals Ltd

INVESTIGATION OF THE HETEROGENEITY OF RHESUS MONKEY ALPHA1-ANTITRYPSIN

RONALD W. BERNINGER and MARIA F. TEIXEIRA Pulmonary Division, Department of Pediatrics, New England Medical Center Hospitals Inc., Tufts

University School of Medicine, Box 208, Boston, MA 0211 l, USA (Tel: 617-956-5240)

(Received 20 January 1986)

Abstract--1. Rhesus monkey alpha~-antitrypsin (n = 144) was examined for heterogeneity by acid starch gel electrophoresis, isoelectric focusing in agarose and agarose gel electrophoresis.

2. In contrast to other studies, no heterogeneity of Rhesus monkey alphat-antitrypsin could be documented using specific antisera.

3. Rhesus monkey alphal-antitrypsin contained a reactive thiol. 4. The pls of the major isoforms of Rhesus monkey alpha~-antitrypsin were 4.63, 4.69, 4.84 and 4.86

at 4°C. 5. No deficiency state of Rhesus monkey alpha~-antitrypsin was detected. 6. The six protease inhibitors in Rhesus monkey sera cross-reacted with antisera to the six human

protease inhibitors.

INTRODUCTION

An animal model for the deficiency state of alpha~-antitrypsin would be of great importance in understanding liver disease in children and/or lung disease in adults. Furthermore, such an animal model would allow evaluation of drug therapies and manip- ulations of the animal model which is not possible in human subjects. Since it had been reported by others (Omoto et al., 1970) that heterogeneity of Rhesus monkey (Macaca mulatta) alphal-antitrypsin exists (n = 40) and an earlier study of Rhesus monkeys (n = 28) found no evidence of heterogeneity of alphal-antitrypsin (Berninger and Mathis, 1976a), we studied a larger populat ion of Rhesus monkeys using several electrophoretic methods. In these current studies, Rhesus monkey alphal-antitrypsin was compared to the PI M phenotyp¢ of human alpha~-antitrypsin by acid starch gel electrophoresis, analytical isoelectric focusing in agarose and agarose gel electrophoresis. Furthermore, purification of monkey alpha~-antitrypsin by thiol disulfide interchange chromatography verified that monkey alphat-antitrypsin contains a reactive thiol like human alpharant i t rypsin. Even though better resolution was obtained in these current studies, we still found no evidence for genetic heterogeneity or a deficiency state of alpha~-antitrypsin in Rhesus monkeys.

We speculate that a severe deficiency of alphal-antitrypsin in wild monkeys may be incompatible with life. Therefore, it is unlikely that a deficiency of alpha~-antitrypsin in wild monkeys can be documented. There is the possibility that a deficiency of alpha~-antitrypsin may be documented in monkeys bred in captivity.

MATERIALS AND METHODS

Materials used in this study were obtained as listed below: substance--company; sodium acetate, glacial acetic acid, sodium azide, concentrated hydrochloric acid, citric acid,

TRIS, boric acid, calcium lactate, sodium barbital, glycine and phosphoric acid--Fisher Scientific Company, Medford, MA; columns, adaptors and DEAE-Sephadex A-50 (lot # 2252)---Pharmacia, Piscataway, N J; ultra pure ammo- nium sulfate---Schwarz/Mann, Orangeburg, NY; refrig- erated centrifuges (Sorvall RT 6000 and Superspeed RC5B)--Dupont, Newtown, CT; sodium hydroxide, barbi- turic acid--Mallinchrodt, St Louis, MO; iodoacetamide-- Polysciences; dithiothreitol (DTT)--Bio-Rad Laboratories, Richmond, CA; cellulose acetate strips--Gelman Sciences, Inc.; methanol--Baker Chemical Co., Phillipsburg, NJ; ethanol--New England Medical Center Hospital Phar- macy, Boston, MA; rabbit anti-monkey serum--Micro- biological Associates, Bethesda, MD; rabbit anti-human alpharantitrypsin--Kallestad Laboratories, Inc., Austin, TX.

Subjects Healthy Rhesus monkeys (2.8-4.1 kg) environmentally

conditioned (animals maintained under stable physical conditions, dietary conditions, with treatment for bacterial and parasitic diseases, and acclimated to handling procedures) for 2 months to 2.5 years were bled by venipuncture at the US Army Medical Research Institute of Infectious Diseases. Blood was allowed to clot for 2 hr at room temperature and separated serum was stored at -70°C.

Isolation of Rhesus monkey alpha~-antitrypsin Rhesus monkey alpha I-antitrypsin was isolated by ammo-

nium sulfate precipitation, ion exchange chromatography using DEAE-cellulose, and affinity chromatography on Concanavalin A-Sepharose as described earlier (Berninger and Mathis, 1976). Thiol disulfide interchange chromatography on activated Thiol-Sepharose was used to remove traces of Concanavalin A (Berninger and Talamo, 1979) and to further purify the monkey alpharantitrypsin. Isolated monkey alpha~-antitrypsin was characterized by immunoelectrophoresis (Grabar and Williams, 1953), Ouch- terlony (Ouchterlony, 1949) and SDS polyacrylamide gel electrophoresis (Weber et al., 1972). Monkey serum and isolated alphal-antitrypsin were tested against antisera to whole monkey serum and antisera (Behring Diagnostics) to the following human proteins: alpha~-antitrypsin, alphat-lipoprotein, alphas-acid glycoprotein, transferrin,

325

326 RONALD W. BERNINGER and MARIA F. TEIXEIRA

albumin, Gc globulin, alpha~-antichymotrypsin, inter- alpha-trypsin inhibitor, antithrombin III, Cls inactivator, alpha2-macroglobulin, IgG, IgA and IgM.

Production of antisera to Rhesus monkey alphal-antitrypsin

Production of antisera to lyophilized Rhesus monkey alpha~-antitrypsin was initiated in one rabbit, as outlined earlier (Berninger and Mathis, 1976a). The IgG and IgA were isolated by the method of Harboe and Ingild (1973) using an LKB (Rockville, MD) MultiRac, Peristaltic Micro- perpex pump and Uvicord S detector at 280 nm. The eluate was dialyzed using tubing from Spectrum Medical Indus- tries (MW cut off 12,000-14,000) and subsequently concentrated with Immersible Molecular Separators (NMWL 10,000; Millipore, Bedford, MA).

Isolated antibody to monkey alpharantitrypsin was examined by immunoelectrophoresis (Grabar and Williams, 1953) and Ouchterlony methods (Ouchterlony, 1949) using 1% (w/v) Seakem Agarose (lot # 6365, Marine Colloids, Rockland, ME) in Tris/barbital/sodium barbital buffer, pH 8.8 (Gelman, Ann Arbor, MI). Both native monkey serum and isolated monkey alphat-antitrypsin were tested against the antibody to Rhesus monkey alpha~-antitrypsin.

PI typing

Utilizing sera, electrophoretic mobilities and patterns for monkey alpha~-antitrypsin were compared to human alpha~-antitrypsin by acid starch gel electrophoresis, isoelectric focusing in agarose and agarose gel electrophoresis. Previously published procedures were followed with some modifications. These are briefly discussed for each technique.

A 13 % starch gel was prepared by dissolving 7 g of freshly hydrolyzed (Fagerhol, 1968) potato starch (lot # 792325, Fisher, Fair Lawn, NJ) and 19 g of hydrolyzed starch (lot #365-1, Connaught Laboratories, Willowdale, Ontario) in 200ml of diluted stock buffer (10ml to 200ml with deionized water), pH 5.03. Electrophoresis (Talamo et al., 1978b) was performed at 35 mA (constant current) for 1 hr, then at 1000 V (constant voltage) for the rest of the run on a cooled chamber maintained at 4°C by a multitemp water circulator (LKB). Gels were then either stained for protein in a 0.25% Coomassie Blue solution (Sigma, St Louis, MO), immunofixed (Arnaud et al., 1977b), or used as the origin material for crossed electrophoresis. Starch strips containing alpha~-antitrypsin bands were subjected to electrophoresis at 4°C, 500 V for 5 hr (Laurell, 1965; Talamo et al., 1978b) in a 1% (w/v) Seakem agarose gel containing 0.2% (v/v) antibody to alpha~-antitrypsin.

The PI patterns of human and monkey alpha~-antitrypsins were compared by isoelectric focusing in agarose (Qureshi and Punnett, 1982). Agarose-EF (LKB, batch # 000001) was dissolved in deionized water containing sorbitol (Fisher), ACES (Polysciences, Warrington, PA) and serine (Sigma). Carrier Ampholines, pH 3.5-5, pH 445 and pH 5-8 (batch # s 16, 35 and 10, respectively, LKB) were added at 70°C and degassed. The agarose mixture was poured onto the hydrophilic side of a polyester plate (Gel Bond, Marine Colloids) using the 0.5 mm capillary gel casting kit available from LKB. Samples were reduced with DTT and alkylated with iodoacetamide (Qureshi and Punnett, 1982) and 10#1 pipetted onto sample pads (5 x I0 mm, LKB). Isoelectric focusing was performed at 4°C using an LKB Ultrophor 2117 and 2103 power supply. Power settings were adjusted relative to length of the gel (Qureshi and Punnett, 1982). The pH gradient was measured by eluting sections of the gel in degassed, deionized water. After focusing, the fixed gels were either dried and stained for protein with 0.5% Coomassie Blue solution or immunofixed (Arnaud et al., 1977b) at room temperature.

In order to compare relative mobilities, 1% Seakem agarose gels were prepared in Laurell's buffer, pH 8.6 for agarose gel electrophoresis (Johansson, 1972). Thirty-

seven ml of hot agarose were poured onto a level hot glass plate (1 mm x 26 cm x 12.5 cm, LKB) and a slit forming device (Med. Eng. Dept., MGH, Boston, MA) positioned immediately. Electrophoretic wicks (Orion Diagnostics, Helsinki, Finland) were positioned along anodal and cath- odal ends of the gel.

Trypsin inhibitory capacity

The trypsin inhibitory capacity (TIC) of Rhesus monkey sera was measured by the method of Eriksson (1965) outlined in Talamo et al. (1978a). The same lot of trypsin was used for all sera. Results were expressed as mg trypsin inhibited/ml of sample.

RESULTS Subjects

Sera from a total of 144 Rhesus monkeys were analyzed.

Isolated alpha~-antitrypsin

The isolated Rhesus monkey alphal-antitrypsin exhibited 1 arc when tested by immunoelectrophoresis against both the antisera to Rhesus monkey serum and the prepared antisera to Rhesus monkey alphal-antitrypsin. Furthermore, there was complete fusion of the arcs from serum alpha~-antitrypsin with the isolated alphal-antitrypsin when tested against antibody to either whole Rhesus monkey sera or antibody to isolated monkey alpha~-antitrypsin. Ouchterlony double diffusion experiments in which the isolated Rhesus monkey alphal-antitrypsin was diluted and tested against the antibody to whole Rhesus monkey antisera, and in which the antisera was diluted and tested against a constant amount of isolated Rhesus monkey alphal-antitrypsin, all exhibited 1 arc with complete fusion in more concentrated samples and no arcs in diluted samples. Similar experiments with the antisera to Rhesus monkey alphal-antitrypsin exhibited 1 arc. Precipitin lines were found between monkey serum and antisera to all six human protease inhibitors (aiphaj-antitrypsin, alpha,-antichymotrypsin, inter-alpha-trypsin inhibitor, ant i thrombin III, C l s inactivator and alphaE-macroglobulin) and alphal-acid glycoprotein. Only one precipitin line was formed between isolated monkey alpha~-antitrypsin and antiserum to human alpha~-antitrypsin. No precipitin lines formed when tested against antisera to the human proteins listed in the Materials and Methods.

The isolated Rhesus monkey alphal-antitrypsin exhibited 1 band, even in overloaded gels, when tested by SDS polyacrylamide gel electrophoresis. The data from these experiments are not shown since they are identical with those obtained in previous studies of purified Rhesus monkey alpha~-antitrypsin (Berninger and Mathis, 1976a).

P I typing results

It was found by trial and error that an adjustment of the starch gel buffer to pH 5.03 allowed better resolution of the alpha~-antitrypsin banding patterns visualized by protein staining. The use of the more basic buffer, compared to pH 4.95 in our previous studies (Berninger and Mathis, 1976a) and those of others (Omoto et al., 1970), also allowed for a clearer background in the alpha~-antitrypsin region of the starch.

The heterogeneity of Rhesus monkey alphal-antitrypsin 327

PI pattern appeared cleaner and sharper following reduction of sera. Therefore, reduction and alkylation were performed in all isoelectric focusing studies. The results of comparing the pattern with the extra bands and those without the extra bands by protein staining and isoelectric focusing are shown in Fig. 4. Com- parison of the two patterns followed by immunofixation are shown in Fig. 5. The pls of M 2, 4 and 6 were 4.54, 4.60 and 4.64, respectively, while the pls of the monkey alphal-antitrypsin were 4.63, 4.69, 4.84 and 4.86. The pls were determined at 4°C.

In order to compare the mobility of the two different types of the monkey alphal-antitrypsin with the PI M and PI Z phenotypes of human alphar-antitrypsin, an agarose gel electrophoresis was used. The results of immunofixation in agarose for a total of 8 monkeys without the extra bands and 6 monkeys with the extra bands are shown in Fig. 6.

Trypsin inhibitory capacity The 138 monkeys which did not have the extra

bands had a TIC mean + SD of 1.48 + 0.32 (range 0.88-2.73) while the 6 monkeys with the extra bands exhibited a TIC me a n+ SD of 1.37 +0.18 (range 1.11-1.64).

I 2 3 4 5

Fig. 1. Comparison of human and Rhesus monkey (Macaca mulatta) alphaFantitrypsin by acid starch gel electro° phoresis, pH 5.03. Anode at top and cathode at bottom. The gel is stained for protein (Coomassie Blue). Tracks l 3 show examples of the extra bands (black dots) in 6 monkey samples compared to an example of 138 monkeys without the extra bands in track 4. Track 5 contains human PI M

serum. Human PI M6 is marked by a triangle.

A total of 144 Rhesus monkeys were screened by acid starch gel electrophoresis, followed by a protein stain. A total of 6 monkeys out of the 144 exhibited the pattern shown in the first 3 wells of Fig. 1 and contain 2 extra bands relative to the pattern found in well 4 in which these 2 bands are missing (Fig. 1). However, examination of these patterns obtained in acid starch electrophoresis followed by immunofixation (Fig. 2) indicated that there was no difference between the two types of patterns for 22 monkeys without the extra bands and the 6 monkeys with the extra bands. Furthermore, a sample of 28 different monkeys, other than the ones examined by immunofixation in starch, and the 6 monkeys with extra bands were subjected to crossed electrophoresis after acid starch gel electrophoresis. The same pattern was obtained (Figs 3A and 3B) and were compared with PI M and PI Z (Figs 3C and 3D, respectively).

A total of 21 monkeys without the extra bands and the 6 monkeys with the extra bands were examined by isoelectric focusing in agarose by both protein staining and immunofixation. As noted by others for human sera (Frants et al., 1978, Jeppsson and Franz6n, 1982) and ourselves, the alphal-antitrypsin

l 2 3 4 5

Fig. 2. Immunofixation (with specific antisera) of human and Rhesus monkey (Macaca mulatta) alphaFantitrypsin in acid starch gel eleetrophoresis, pH 5.03. Anode at the top, cathode at the bottom. Tracks 1 and 2 have examples (out of 6) of monkey sera with 2 extra protein bands; track 3 has an example of the pattern obtained for 138 monkeys which did not have the extra protein bands; track 4 has human PI M; and track 5 has human PI Z. No extra alphaFantitrypsin

bands are seen in tracks 1 and 2 compared to track 3.


Fig. 3 A and B. For legend see opposite.

DISCUSSION Initial examination of 144 sera from Rhesus mon-

keys indicated that 6 monkeys might be hetero- zygotes, similar to the PI MZ phenotype in humans. However, upon further investigation with specific antisera to Rhesus monkey alphal-antitrypsin, this was not the case. The presence of these extra bands is probably due to haptoglobin as described by others (Arnaud et al., 1977a; Fagerhol, 1968).

The results on 144 Rhesus monkeys indicate that the PI type in acid starch gel electrophoresis is probably similar in mobility to the PI types denoted BB by Omoto et al. (1970). However, no homozygous BB PI types were found in Rhesus monkeys by Omoto et al. (1970). They reported 1 (Thailand) and 3 (E. Pakistan) PI BC and 14 (Thailand) and 22 (E. Pakistan) PI CC. The reasons for this discrepancy could be due to the fact that in the current study

we used a higher gel pH (5.03 compared to 4.95) which is known to increase the mobility of the alphal-antitrypsin bands (Talamo et al., 1977b). Fur- ther evidence to support this speculation comes from the observation that in our earlier work (Berninger and Mathis, 1976a) we used a gel pH of 4.95 and obtained a pattern similar to the PI CC pattern for the 28 Rhesus monkeys. Retesting some of these sera at the higher gel pH resulted in the faster moving pattern. In addition, the mobility of monkey PI CC, relative to the human PI M, depicted by Pierce (1976) at pH 5.0 in agarose has the same mobility of the PI pattern we obtained in acid starch gel at pH 5.03 (Figs 1 and 2). Furthermore, Omoto et al. (1970) and Berninger and Mathis (1976a) used commercially hydrolyzed starch, whereas our latest study utilized a mixture of freshly hydrolyzed starch and commercially hydrolyzed starch for better resolution. One or

The heterogeneity of Rhesus monkey alphal-antitrypsin 329

Fig. 3 C and D.

Fig. 3 A, B, C and D. Crossed immunoelectrophoresis. An acid starch gel electrophoresis was performed in the first dimension (anode left, cathode right) and a crossed immunoelectrophoresis was performed in the second dimension in agarose (anode top, cathode bottom) containing rabbit anti-monkey alphacantitrypsin (Fig. 3A and 3B) or rabbit anti-human alphal-antitrypsin (Fig. 3C and 3D). Figure 3A did not contain the extra protein bands shown in Fig. I, while Fig. 3B did contain the 2 extra protein bands shown in Fig. I. Figure 3C is human PI M while Fig. 3D is human PI Z. The dot marks the position

of human PI M6.

more of these factors could differentially effect the mobility of the Rhesus monkey alpha~-antitrypsin. The true mobility of Rhesus monkey alpha~- antitrypsin, determined by isoelectric focusing in agarose (Figs 4 and 5), is slower than the PI AA but faster than the PI BB phenotypes outlined by Omoto et al. (1970) in an acid starch gel. By agarose gel electrophoresis at pH 8.6, Rhesus monkey alpha~-antitrypsin is slower than human PI M with similar mobility to human PI Z (Fig. 6).

We find no evidence of phenotypic heterogeneity in Rhesus monkey alpha~-antitrypsin using acid starch gel electrophoresis, agarose isoelectric focusing or agarose gel electrophoresis. The results are in contrast to those described by Omoto et al. (1970) using acid starch gel electrophoresis and protein staining but in agreement with our earlier smaller (n = 28) study (Berninger and Mathis, 1976a). The heterogeneity described by Omoto et al. (1970) existed in the major bands of alpha~-antitrypsin but specific anti-


I 2 5 4 5 6 7 Fig. 4. lsoelectric focusing in agarose gel. Anode top, cathode bottom. The dried gel was stained for protein (Coomassie Blue). Tracks 1, 3 and 5 contain examples of monkey sera which do not have extra protein bands in Fig. 1; tracks 2, 4 and 6 contain examples of 6 monkeys which did have 2 extra protein

bands in Fig. 1. Track 7 has a human PI M with PI M6 shown by a triangle.

I 2 5 4 5 6 7

Fig. 5. Immunofixation of alpha~-antitrypsin after isoelectric focusing in agarose. The dried gel was stained for protein (Coomassie Blue). Track 1 contains human PI M sera with PI M6 shown by a triangle. Tracks 2, 4 and 6 contain monkey samples which do not contain the extra protein bands in Fig. 1; tracks 3, 5 and 7 contain monkey samples which do have extra protein bands shown in Fig. 1. The major monkey

isoforms with pls 4.63, 4.69, 4.84 and 4.86 at 4°C are shown by dark squares.

The heterogeneity of Rhesus

I 2 5 4 5

Fig. 6. Immunofixation of human and monkey alpharantitrypsin in agarose gel. Anode top, cathode bottom. Tracks 1 and 5--human PI Z; track 2--human PI M; track 3--Rhesus monkey serum with extra protein bands in Fig. 1; track 4--Rhesus monkey serum which does not have

extra protein bands in Fig. 1

sera was not used to examine the heterogeneity further. Since we do not know the source of our monkeys caught in the wild, it is possible that no heterogeneity existed in our particular monkey popu- lation (144 samples in this study, 28 samples in an earlier study). Heterogeneity in alphal-antitrypsin has been detected in a variety of monkey species (Kueppers and Ganesan, 1977; Pierce, 1976; McDer- mid and Ananthakrishnan, 1972; Martin et al., 1976, Omoto et al., 1970). However, Pierce (1976) found no heterogeneity in alpha~-antitrypsin in 116 baboons while Martin et al. (1976) detected heterogeneity in 27 baboons.

The lowest TIC detected in our studies was 0.88 mg per ml compared to a group mean of 1.48. However, these monkeys had the same type of alpha~- antitrypsin as the rest of the monkeys in the group tested. The TIC of monkey sera is quite high, probably due to stress, until they become accustomed to human handlers and surroundings (Berninger and Mathis, 1976b). We speculate that the severe deficiency of alpha~-antitrypsin seen in humans may be incompatible with life in the wild for monkeys. It is possible that such a severe deficiency of alphal-antitrypsin could be detected in monkeys bred

monkey alphal-antitrypsin 331

in captivity. A severe deficiency of alphal-antitrypsin has not been documented for any of the monkey species.

While these studies did not detect a heterogeneity of Rhesus monkey alphal-antitrypsin, we did deter- mine several properties of the Rhesus monkey alphal-antitrypsin molecule and monkey protease inhibitors not previously reported. Reduction of the alphal-antitrypsin by DTT resulted in a cleaner pattern of monkey alphal-antitrypsin as noted by others for human aipha~-antitrypsin. We have also shown that it is possible to isolate the Rhesus monkey alpha~-antitrypsin by thiol disulfide interchange chromatography which indicates that there is a reactive thiol in the Rhesus monkey alpha,-antitrypsin similar to that found in the human alpha~-antitrypsin (Lau- rell et al., 1975), baboon monkey alphal-antitrypsin (Pierce, 1976) and several other monkey alpha l- antitrypsins (Pierce, 1976). By increasing the pH of the acid starch gel to pH 5.03, we were able to improve the separation of the monkey alphal- antitrypsin variant. It is clear that the Rhesus monkey alphal-antitrypsin has a slower mobility by acid starch gel electrophoresis and isoelectric focusing than the human PI M phenotype. However, we were not able to demonstrate any heterogeneity in Rhesus monkey alphal-antitrypsin. Surprisingly, the 6 protease inhibitors, including alphal-antitrypsin, in monkey sera cross-reacted with antibody to the 6 human protease inhibitors.

Acknowledgements--The authors would like to thank Mr Charles Fields for assistance with isolation of the monkey alpha~-antitrypsin and Ms Susan Booker for help with the alpha~-antitrypsin PI typing in starch and Dr Henry L. Dorkin for helpful discussions. The secretarial assistance of Ms Irene Hartford is gratefully acknowledged. These studies were supported in part by the NIH Biomedical Research Support Grant 2, SO7-RRO 5598-19 to New England Medical Center.

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investigation of the heterogeneity of rhesus monkey alpha1-antitrypsin

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