A developmental analysis of the production of chymosin and pepsin in pigs

Comp. Biochem. Physiol. Vol. 68B, pp. 9 to 13

0305-0491/81/0101-0009502.00/0

© Pergamon Press Lid 1981. Printed in Great Britain

A DEVELOPMENTAL ANALYSIS OF THE P R O D U C T I O N OF CHYMOSIN A N D PEPSIN IN PIGS BENT FOLTMANNt, ARNE L. JENSEN1, PETER LONBLAD1, ERIK SMIDT1 and NILS H. AXELSEN2 1Institute of Biochemical Genetics, University of Copenhagen, Denmark and 2Immunochemical Section, Treponematosis Dept., Statens Seruminstitut, Copenhagen, Denmark

(Received 18 April 1980) Abstract--1. Calculated as active enzymes the gastric mucosa of piglets contains 1-4 mg of chymosin

(EC 3.4.23.4) per g of mucosa during the first week of life. No chymosin is present in pigs older than 2 months. 2. The production of pepsin (EC 3.4.23.1) is very low during the first week of life; a rapid increase occurs after about 3 weeks. 3. Pig chymosin and pig pepsin have higher milk clotting activity against porcine milk than against bovine milk. 4. Neonatal proteinases with high milk clotting and low general proteolytic activities are probably important for postnatal uptake of immunoglobulins.

INTRODUCTION It is well-known that the gastric juice of calf (Bos taurus) contains a proteinase (chymosin; EC 3.4.23.4) with high milk clotting and low general proteolytic activity (review by Foltmann, 1966). However, relatively few investigations have been carried out on neonatal gastric proteinases from other mammals. Earlier analyses on development of proteolytic enzymes from the stomach of pig (Sus scrofa) have mainly relied on the general proteolytic activity (Cranwell & Titchen, 1976; Cranwell, 1977; Decuypere et al., 1978), and these investigations showed little or no proteolytic activity during the pigs first week of life. In a previous paper (Foltmann et al., 1978) we have reported that stomach of newborn pig contains a proteinase that is different from the gastric proteinases of the adult pig. We also found that the proteinase from newborn pigs is immunologically related to calf chymosin, and hence the name pig chymosin was introduced. In the investigations reported in the present paper the amounts of pig chymosin and pig pepsin A (EC 3.4.23.1) have been determined with quantitative immunoelectrophoresis. The results show a decline of chymosin production after 5 to 10 days of age, and we are able to confirm the earlier observations that a rapid increase in pepsin production takes place 2 to 4 weeks after birth of the pig. Further we have compared the milk clotting activities of calf chymosin and pig gastric proteinases against bovine and porcine milk. The results suggest an adaptation between the proteolytic specificities of the gastric proteinases and the primary structures of the caseins. MATERIALS AND METHODS

Animals and extractions Fetuses of pigs were obtained from pregnant sows brought to the slaughter house FSA, Ringsted. The fetuses

were stored frozen until dissection took place at Statens Seruminstitut, Copenhagen. The age of fetuses was estimated from their weights and development of teeth and hairs. Stomachs from 15 normal healthy pigs of age from 1 to 17 days were removed just after the animals were slaughtered. In addition pig stomachs were collected from pigs that died in the sty from accidents or disease. The stomachs were removed as soon as death was observed. Only 40 stomachs that looked healthy by macroscopic examination were used. All stomachs were flushed with cold water and stored at -20°C before use. Each stomach was extracted separately. The mucosa was scraped off with a scalpel, and extraction was carried out with 5 ml of water per g mucosa. After treatment for 3 min in a Potter-Elvehjem homogenizer, suspended tissue was removed by centrifugation at 12,000 g for 15 min at 2°C. Most of the extracts from stomachs of piglets contained both prochymosin and active chymosin; before assay of enzymatic activity or immunological analyses the zymogens were converted into active enzymes by lowering pH to 2 for 30min. The results are expressed directly as mg of enzyme/ml of extract. If we assume an even distribution of enzyme in the liquid phase after homogenization, the results may be converted into an approximate value of mg/g mucosa by multiplying by 6.

Pure enzymes Crystalline pig pepsinogen A was from Worthington (Freehold, N J, U.S.A.). Crystalline pig pepsin A was from Sigma (St Louis, MO, U.S.A.), the concentrations were determined using the absorbances given by Arnon & Perlmann (1963), A~m,27a 12.5 and 14.5 respectively. Crystalline calf chymosin (A~m,27 a 14.3) was prepared as described earlier (Foltmann, 1966). Pig chymosin was prepared by chromatographic fractionations similar to those used for preparation of calf chymosin (Foltmann, 1970). A detailed examination of pig chymosin is in progress; the enzyme appears to have great similarity to calf chymosin, thus the concentration of pig chymosin was tentatively determined using Alc~,2"~a 1% 14.3.

Antisera and immunoassay Antisera against chromatographically purified pig chymosin were raised in rabbits using the immunization procedures of Harboe & Ingild (1973). The antisera against pig

BENT FOLTMANN et al.

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Fig. 1. Developmental change in potential content of chymosin in gastric mucosa li-om pigs. Determinations by rocket immunoelectrophoresis after prochymosin has been converted into chymosin. The results are expressed as mg of chymosin/ml of extract of gastric mucosa. The vertical bars indicate the scattering and the numbers of animals in each age group are given above the lines. Circles indicate average of each group. The age before birth is estimated + 10 days. pepsins A and C were obtained from Chr. Hansens Laboratory, Copenhagen, Denmark. Rocket immunoelectrophoresis was carried out as described by Weeke (1973). The standards were crystalline porcine pepsinogen A that was activated at pH 2 before use, and chromatographically purified pig chymosin. In our experiments the solutions of pig chymosin turned to be less stable than solutions of calf chymosin; after 6 months at -20°C the milk clotting activity of a solution of chromatographically purified pig chymosin was reduced by approx 30%. Milk clotting assay For routine determinations of milk clotting activity each assay was performed with 10ml of reconstituted bovine skim milk pH 6.3 and l ml of enzyme solution. The assay was carried out in bifurcated glass tubes, and the activity is expressed in chymosin units (CU) as described previously (Foltmann, 1970). F6z~ clotting tests with porcine milk, the milk was skimmed by centrifugation at 6000 g for l0 min at 5°C and a solid layer of milk fat was removed. The acidity of the milks varied from pH 6.7 to 7.2 and was adjusted to 6.3 with 0.2 M HC1. The clotting experiments with porcine milk were carried out with 5 ml of milk and 0.5 ml of enzyme solution. Parallel experiments were carried out with 5 ml of reconstituted bovine milk and the results are expressed as the amount of enzyme that would clot 5 ml of milk in 5 min at 30°C. RESULTS A N D DISCUSSION

Immunoelectrophoretic determinations The development of chymosin production in pigs is illustrated in Fig. 1. Large variations were found in

the contents of enzymes from the stomachs of different animals of the same age. Of the 15 fetuses with an age of ca. 20 days before birth, 13 belonging to one litter had an average concentration of 0.58 mg of chymosin/ml in the extracts and a range of 0.16-0.90 mg/ml. We also found that the enzyme contents of stomachs from slaughtered animals scattered the same way as those obtained from animals that died from accident or disease. Consequently, we consider that all our observations are typical for the development of production of chymosin in pigs. Immunoassays are very useful tools, but their application for quantification of gastric proteinases requires a critical discussion. Our monospecific antisera were raised in rabbits immunized with active enzymes, and the rocket immunoelectrophoreses were carried out at pH 8.6. The gastric proteinases will normally lose their activities at pH above 7; thus both deposition of the immunogens in the animals and the immunoelectrophoretic analyses took place under circumstances that lead to denaturation of enzymes as measured by their enzymic activities. It is therefore possible that our antisera, in addition to the active enzymes, also precipitate more or less denatured components. This will influence the apparent activity per mg of enzyme, but we were looking for the production of enzyme and hence a denaturation during the immunoassay did not impair the developmental study. As regards the presence of zymogens we have 3

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Fig. 2. Developmental change in potential content of pepsin A in gastric mucosa of pigs. Determinations by rocket immunoelectrophoresis after pepsinogen has been converted into pepsin. The results are presented as in Fig. 1.

Development of chymosin and pepsin observed that with the antisera used in this investigation the zymogens gave weaker precipitates that were easily distinguished from the sharp and dense precipitates given by the extracts after activation at pH 2. In an evaluation of the quantitation it should also be recalled that, measured by milk clotting activities, the standard solutions of chromatographically purified pig chymosin were less stable than solutions of calf chymosin, and we estimate that the inaccuracy of the individual immunochemical determinations of pig chymosin was about + 30~o. In spite of these critical comments the conclusion that appears from Fig. 1 is clear-cut. Prochymosin is present in the fetal gastric mucosa from about three weeks before birth. Calculated as mg chymosin/g mucosa the following average values were found after birth: 0-6 days: 2.3; 7-12 days: 0.9; 18-35 days: less than 0.5; older than 2 months: chymosin is absent. The immunoelectrophoretic determinations of pepsin suffer from the same inherent problems as discussed above, but the results that are shown in Fig. 2 are very convincing. Measured as pepsin, pepsinogen is virtually absent in mucosa from piglets that are younger than 5 days. Only three stomachs from this age group showed pepsinogen concentrations of about 0.05 mg of pepsin/g mucosa, and the typical amounts were so small that we did not obtain measurable rockets. The production of pepsinogen increased after about one week of life with a rapid increase after about 3 weeks. This is consistent with earlier observations on the development of proteinases in the stomach of pigs (Cranwell & Titchen, 1976; Cranwell, 1977; Decuypere et al., 1978). In addition to the predominant pepsin A the gastric juice of pigs contains minor components, designated pepsin B (EC 3.4.23.2), C (EC 3.4.23.3), and D (Ryle, 1970). Of these pepsin D is probably unphosphorylated pepsin A, and the two components are determined together in the immunological assay. Only little is known about pepsin B, and we have made no attempt to analyse for the content of pepsin B. Pepsin C apparently corresponds to human gastricsin (Tang, 1970) also called human group II pepsin (Samloff, 1971). We have only had a limited supply of antisera against porcine pepsin C and we have had no standard for quantitative determinations. Ten stomachs of pigs have been analysed for the content of

11

pepsin C measured relative to the content of pepsin C in the stomach of a 6 month old pig. The results indicate that in some animals the production of pepsin C develops earlier than pepsin A. Considering the large individual variations in the production of chymosin and pepsin these few observations do not allow a definitive statement about the development of production of pepsin C. Enzymic activity

The enzymic activities of the extracts from pig stomachs were assayed by clotting of reconstituted bovine skim milk. The results shown in Fig. 3 illustrate that the milk clotting activity follows the content of enzymes as determined by immunoelectrophoresis. In extracts that contain chymosin together with larger amounts of other gastric proteinases we have not tried to evaluate the contribution to the milk clotting activity of the individual components, but up to 5 days of age nearly all milk clotting activity was derived from the content of chymosin. In such extracts we have observed a systematic discrepancy between the milk clotting activity per mg of chymosin determined by immunoelectrophoresis and the milk clotting activity per mg of pig chymosin just after purification by chromatography. The chromatographically purified chymosin exhibited a specific activity of ca. 25 CU/mg, while after activation at pH 2 the crude extracts on the average had a specific activity of ca. 15 CU/mg of chymosin as determined by rocket immunoelectrophoresis. This discrepancy is not fully elucidated, but two factors may contribute to an explanation. First, as pointed out above, partly denatured proteins in the crude extracts may contribute to the height of the rockets. Second, the activation segment or propart peptides that are liberated during the limited proteolysis of the zymogen may have some inhibitory effect as it is found in the activation of porcine pepsinogen (Herriott, 1939). Clotting of bovine milk is often used as an assay for proteolytic enzymes, but one may argue that it is not correct to assay the clotting enzymes from one species with milk from another. We have therefore also tested milk clotting activity of extracts from pig stomachs against porcine milk. Experience from dairy research has shown that the clotting ability of milk from individual cows often

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Fig. 3. Milk clotting activity of extracts from stomach of pigs after 30 min activation at pH 2. The results are expressed in chymosin units CU/ml of extract. Scattering and averages are presented as in Fig. 1; (the milk clotting activity of 13 Stomachs from one litter of 13 fetuses (20 days before birth) were only tested in the mixed extracts, the scattering of this litter is therefore not expressed in the milk clotting activity).

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BENT FOLTMANNet al. Table 1. Clotting of bovine and porcine milk with pig and calf proteinases (The results are expressed as #g of enzyme that clots 5 ml of milk in 5 min at 30°C) Reconstituted bovine skim milk Pig chymosin (in crude extract of piglet stomachs) Pig pepsin A Calf chymosin

16.8 9.70 1.69

differs greatly. The clotting ability of porcine milk varied also from sow to sow, but all results pointed in the same direction. In our first experiments five samples of porcine milk were received on different days. With all samples of porcine milk, pig chymosin showed a milk clotting activity that was 4--10 times higher than the clotting activity against bovine milk. In the final experiments three samples of porcine milk were tested with extract of piglet stomachs, in parallel with solutions of pig pepsin and calf chymosin. The concentrations of pig chymosin in the crude extracts were determined by rocket immunoelectrophoreses. As mentioned above it may have been overestimated, but since the same solution was used in all experiments, a minor error in the absolute concentration does not affect the relative milk clotting activity. Each solution of enzyme was tested in at least three different concentrations against each sample of milk. By plotting the clotting times against the reciprocal value of the amounts of enzymes, linear graphs were obtained, and the amount of enzyme required to clot 5 ml of milk was determined (Table 1). With the milk samples used in these experiments pig chymosin has a clotting activity against porcine milk that is 6-8 times that against bovine milk, pig pepsin has also a higher milk clotting activity against porcine milk than against bovine milk, while calf chymosin has less activity when tested with porcine milk. Comparison between the clotting activities of pig chymosin and pig pepsin shows that the latter has greater activity against bovine milk. This is presumably due to the much larger unspecific proteolytic activity of pepsin. Our knowledge about the specificity requirements of calf chymosin and the structure of the caseins may contribute to explain the differences between the two chymosins. During the clotting of bovine milk a C-terminal glycopeptide is cleaved from x-casein through a limited proteolysis of a Phe-Met bond. Using synthetic model peptides Visser et al. (1977) found that methionine may be substituted by norleucine, but the activity of calf chymosin is reduced if methionine is substituted by leucine. This indicates that an amino acid with a branched side-chain does not fit in the binding site of calf chymosin. We do not know the entire amino acid sequence of porcine x-casein, but it has been reported that the glycopeptide has N-terminal isoleucine (Mercier et al., 1976), and this is consistent with reduced activity of calf chymosin against porcine milk. It is known that both chymosin and pepsin have extended binding sites (Visser et al., 1977; Fruton, 1976) and the amino acid residues preceding the bond that is cleaved may also contribute to the specificity.

Porcine skim milk days after farrowing 14 18 21 2.22

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However, our results suggest an adaptation of the binding sites of both chymosins and pepsins to the primary structures of the x-caseins. Evolutionary and physiological aspects

In a recent review (Foltmann & Axelsen, 1980) we have pointed out that the predominant pepsinogens from gastric mucosa of dog, cat, horse, pig, cow and man all are immunologically related. Furthermore antibodies against calf chymosin precipitated components in extracts from gastric mucosa of dog, cat and pig, but none of these components reacted with antipepsin sera. These observations are consistent with the available information about primary structures (Foltmann & Pedersen, 1976). Calf chymosin and bovine pepsin have about 50Y/o of identity in their primary structures, while bovine and porcine pepsin have about 75~o of identity in their primary structures. All these observations indicate that separate genes for pepsin A and for chymosin were present before divergence of the different lines of mammals. With these considerations in mind it is noteworthy that the milk clotting experiments (Table i) suggest that in spite of the inter-species resemblance of pepsins and chymosins respectively, the two enzymes from pig show adaptation in substrate specificity for the casein of porcine milk. Concerning the physiological background for the developmental change in production of gastric enzymes we have only limited information. From experiments with calves kept on different diets Garnot et al. (1974) suggested that the production of chymosin was induced by casein. Likewise Cranwell (1977) observed that creep-fed pigs have a greater secretion of pepsin than milk-fed pigs. ' It is most likely that the production of chymosin is related to the digestion of milk proteins, but since pepsin A also has a considerable milk clotting activity it is hardly this activity that is the most important property of chymosin. We know that all the animals in which we have demonstrated anti-chymosin precipitating enzymes also have a postnatal uptake of immunoglobulins from colostrum (review by Brambell, 1970). We also know that at least pig pepsin will cleave immunoglobulins into the F(ab)2 and Fc fragments. This means that large amounts of pepsin would be detrimental for the postnatal uptake of immunoglobulins. A detailed investigation on the specificity of chymosin against immunoglobulins of colostrum is required. However, for the reasons outlined above we suggest that the chymosins represent a group of neonatal proteinases with sufficient milk clotting activity, but with

Development of chymosin and pepsin so low general proteolytic activity that extensive degradation of immunoglobulins does not occur in the stomach. The immunoglobulins are further protected against degradation by the pancreatic proteinases through the trypsin inhibitors of the colostrum (Laskowski & Laskowski, 1951; Pedersen et al., 1971). The phYsiological significance of the formation of the casein-clot and the final uptake of immunoglobulins are not yet elucidated in details. Leary & Lecce (1979) have discussed the problem about uptake of irnmunoglobulins by the small intestine of the neonatal piglet. Their own experiments suggest that the uptake occurs by micropinocytotic activity of the enterocytes in the proximal part of the small intestines. This activity was found to be stimulated by other macromolecules like serum albumin or polyvinylpyrrolidone. The effect of casein was not investigated, but it may well be that one of the functions of the clot is to stimulate the pinocytotic activity and thereby the uptake of immunoglobulins from colostrum. Acknowled#ements--We thank Mr J. Clausen (Lerchenfeld near Kalundborg) and Mr V. Danielsen (The Pig Experiment Station "Sjlelland III" near Roskilde) for delivery of piglet stomachs and samples of porcine milk. We also thank Dr L. Bruun for assistance by dissections of fetuses, and Ms T. Dannemann Jensen for assistance in performing the immunological analyses.

REFERENCES

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FoL'rMANN B. (1970) Prochymosin and chymosin (prorennin and rennin). Meth. Enzym. 19, 421-436. FOLTMANNB. & AXELSENN. H. (1980) Gastric proteinases and their zymogens. Phylogenetic and developmental aspects. In Enzyme Regulation and Mechanism of Action (Edited by MILDNERP. & Rms B.), pp. 271-280. Pergamon Press, Oxford. FOLTMANN B., I..ONBLAD P. & AXELSEN N. H. (1978) Demonstration of chymosin (EC 3.4.23.4) in the stomach of newborn pig. Biochem. J. 169, 425-427. FOLTblANNB. & PEDERSENV. B. (1977) Comparison of the primary structures of acidic proteinases and of their zymogens. In Acid Proteases: Structure, Function and Biology (Edited by TANG J.) pp. 3-22. Plenum Press, New York. FRUTONJ. S, (1976) The mechanism of the catalytic action of pepsin and related acid proteinases. Adv. Enzym. 44, 1-44. GARNOTP., VALLESE., THAPONJ.-L., TOULLECR., TOMASSONE R. & RIBADEAU-DUIVlASB. (1974) Influence of dietary proteins on rennin and pepsin content of preruminant calf veil. J. Dairy Res. 41, 19-23. HARBOEN. & INGILDA. (1973) Immunization, isolation of immunoglobulins, estimation of antibody titre. Scand. J. Immunol. 2, suppl, l, 161-164. HERRIOTTR. M. (1939) Kinetics of the formation of pepsin from swine pepsinogen and identification of an intermediate compound. J. gen. Physiol. 22, 65-78. LASKOWSKI M. JR & LASKOWSKI M. (1951) Crystalline trypsin inhibitor from colostrum. J. biol. Chem. 190, 563-573. LEARV H. L. & LECCE J. G. (1979) The preferential transport of immunoglobulin G by the small intestine of the neonatal piglet. J. Nutr. 109, 458-466. MERCIERJ.-C., CHOBERTJ.-M. & ADDEOF. (1976) Comparative study of the amino acid sequences of the caseinomacropeptides from seven species. FEBS Lett. 72, 208-214. PEDERSEN V. B., KEIL-DLOUH,6.V. & KEIL B. (1971) On the properties of trypsin inhibitors from human and bovine colostrum. FEBS Lett. 17, 23-26. RYLE A. P. (1970) The porcine pepsins and pepsinogens. Meth. Enzym. 19, 316-336. SAMLOFr I. M. (1971) Progress in gastroenterology. Pepsinogens, pepsins, and pepsin inhibitors. Gastroenterology 60, 586-604. TANG J. (1970) Gastricsin and pepsin. Meth. Enzym. 19, 406-421. VISSER S., v. ROOIJEN P. J., SCHATTENKERK C. & KERLING

K. E. T. (1977) Peptide substrates for chymosin (rennin). Kinetic studies with bovine x-casein-(103-108)-hexapeptide analogues. Biochim. biophys. Acta 481, 171-176. WEEKEB. (1973) Rocket immunoelectrophoresis. Scand. J. Immunol. 2, suppl. 1, 37-46.