Effect of increased oxygen of hydroxyproline synthesis in the cuticle and body wall of Ascaris lumbricoides

467

BIOCHIMICA ET BIOPHYSICA ACTA

BBA

26352

E F F E C T OF INCREASED OXYGEN ON H Y D R O X Y P R O L I N E SYNTHESIS IN T H E CUTICLE AND BODY WALL OF A S C A R I S L U M B R I C O I D E S M I L O S C H V A P I L AND E V A E H R L I C H

Department of Medicine, University of Miami School of Medicine, Miami, Fla. (U.S.A .) (Received F e b r u a r y 23rd, 197 o)

SUMMARY

I. Ascaris lumbricoides injected with [14Clproline were incubated for 24 h in

7 ° % 02 at normal pressure and in air at 1.6 atm abs. 2. In Ascaris incubated at high 02 concentration a substantial increase in collagenous proline hydroxylation occurred (1.6- 3 times) in the cuticle while little effect or perhaps a decrease in hydroxyproline synthesis was observed in the body wall. 3. When incubating Ascaris at four varying concentrations of 02 (o, 5, 20 and 7° %) the hydroxylation of proline (given as ratio specific activity hydroxyproline/specific activity proline) measured in purified cuticle collagen was maximal when the worms were under 7 ° % 02 and 4 times higher than when under N 2. In the case of muscle collagen, hydroxylation was maximal at 20 % 02; 7 ° % 02 was no better than N 2. 4. The results are discussed in terms of two different protocollagen proline hydroxylases; one in the cuticle stimulated by 02 and one in the body wall that is perhaps slightly inhibited by high 02 . Alternatively, the results may be due to differences mainly in the availability of 02 to protocollagen hydroxylase in the two body layers. 5. Data are presented on the distribution of various forms of collagenous hydroxyproline in Ascaris cuticle and body wall.

INTRODUCTION

The concentration of hydroxyproline in the collagen fraction of the cuticle from the A scaris lumbricoides is considerably less than in the muscle region I-8. As hydroxylation of the peptide precursors of collagen in vertebrates is an enzymatically catalyzed reaction involving 02, the question was raised whether the underhydroxylated collagen of the cuticle might be improved by elevating the 02 concentration in the environment. Under the conditions of these experiments, the extent of proline hydroxylation in cuticle was substantially increased by elevating the O 2 concentration of the environment. On the other hand, the synthesis of hydroxyproline in the body wall was not affected by high 02 concentration in the incubation atmosphere. Elevated ambient 02 levels did not influence the incorporation of proline into collagen or noncollagen proteins of the Ascaris. Biochim. Biophys. Acta, 208 (197 o) 467-474

468

M. CHVAPIL, E. EHRLICH

MATERIALS AND METHODS

Collection of the parasites Ascaris were obtained in the local slaughterhouse and collected immediately after extrusion from pig's intestine. Special cale was taken to prevent the parasites from cooling. The nematodes were washed in a 37 ° warm medium and transferred into a fresh medium. All further manipulations with Ascaris were carried out at exactly 37 °. In every sample, individuals of both sexes of the same length (28-30 cm) and weight (6- 7 g) were used in duplicates within one experiment. To decrease the interindividual variability, five Ascaris were pooled in the last experiment.

Incubation and medium The medium of ELLISON4 was slightly modified by dissolving the individual components in o.I M Tris buffer (pH 7.0) and supplementing streptomycin (IOO/~g/ml) and penicillin (i ooo I.U./ml) to prevent bacterial contamination. Two worms were incubated in a large petri dish in 80 ml of medium, aerated with 250 ml of appropriate gas (see tables). The dishes were placed in a desiccator that was flushed with IOOOml of gas. When working at higher pressures than atmospheric, a pressure chamber with controllable temperature was used. Using fresh medium, the two aforementioned procedures were repeated after 12 h of incubation. The incubation was stopped by cooling the worms in cold saline. In the experiment presented in Table V two worms were incubated in 75o-ml screw-cap bottles in 250 ml of medium of ELLISON4 equilibrated for 15 min with pure N2, 5 % 02, air and 7 ° % O 3 at a flow rate of 7ooml/min. The equilibration and incubation atmosphere was renewed every 8 h during the total 24-h incubation period.

Administration of I14Clproline E14C]Proline, uniformly labelled (Amersham, England), with specific activity 270/~C//~mole, was injected in approx. 0.05 ml of saline into the proximal part of Ascaris in the amount of approx. I #C/g of wet weight.

Chemical analysis Ice-cooled Ascaris were dissected and the gut and reproductive system removed. The body wall, including the muscle and hypodermal layer, was stripped off with a spatula. The residual cuticle was washed and frozen in liquid 0 2 and then pulverized. The body wall was homogenized by grinding with sea sand. Three main sets of experiments are presented in this study. In two experiments (see Tables I I I and V) the homogenate of the cuticle or the body wall was extracted first with 5 M NaC1 twice for a total of 24 h at 4 °. This extract was discarded. The total collagen from the residue of the cuticle as well as from the body wall was isolated according to FITCH et al. 5, using repeated extractions with hot 0.3 M trichloroacetic acid. The precipitate, after trichloroacetic acid extraction, was washed with ethanolether (3 : I, v/v) and ether and represented non-collagenous protein. The non-collagenous protein was dried, pulverized, weighed, and then dissolved in Hyamine Io-X (Packard Instiument Co., Downers Grove, Ill., U.S.A.) and the radioactivity measured and related to the mg of protein. The analysis of collagenous proline is given below. In the other experiment, the neutral salt-soluble and insoluble collagen were Biochim. Biophys. Acta, 208 (197 o) 467-474

PROLINE HYDROXYLATION IN ASCARIS

469

isolated (Table IV). Homogenized tissue was extracted twice with o.45 M NaC1 (neutral salt) for 24 h at 4 °. After centrifugation (16000 × g, 20 min), the neutral salt-soluble collagen was further purified b y extraction with hot trichloroacetic acid, according to FITCH et al. 5. After NaC1 extraction the residual tissue was extracted twice with 0.5 % acetic acid (24 h at 4°). The acetic acid extracts were discarded. Insoluble collagen from the residue was extracted with hot trichloroacetic acid. The individual samples of the hot trichloroacetic acid-extracted material were dialysed against cold tap water, evaporated to dryness, and hydrolysed (6 M HCI, 18 h, IOO° in sealed tubes). The proline and hydroxyproline in the hydrolysate were determined b y the method of TROLL AND LINDSLEY6 and STEGEMANN7. The separation of radioactive proline and hydroxyproline was carried out on Dowex 5oW-XS (4° cm × I cm, 200 mesh), using 1.5 M HC1 for hydroxyproline and 3 M HC1 for proline elution. The fractions containing proline or hydroxyproline were pooled, and the amino acid content and radioactivity were determined. All the determinations were done in duplicates. The radioactivity in each of the experiments was measured on a Packard TriCarb scintillation spectlometer with 67 % efficiency. Determination of ATP was used as an index of viability of Ascaiis under varying experimental conditions during the incubation. The ATP determination was carried out by the Boehringer standard ultraviolet assay using whole Ascaris that were pulverized after freezing in liquid 02. RESULTS

The effect of various experimental conditions on A TP synthesis in Ascaris It is well known that Ascaris survive in N 2 atmosphere for several days s. In air atmosphere, no deviation in ATP concentration was observed during 44 h of incubation (Table I). In 98 % 0 2 and at 1.6 atm abs., no change occurred in the ATP concentration after 7 h of incubation. In 98 % 02 at normal atmospheric pressure and at 3 atm abs. no significant change in ATP concentration was seen during 21 h of incubation. However, after 21 h of incubation the concentration of ATP decreased. The contraction of the body wall after the longitudinal dissection had been used as another proof of the viabihty of the Ascaris. In the majority of studies to be reported, a mixture of 7 ° % 0 2 plus 2 % C02 plus 28 % N 2 for 24 h of incubation, and air at 1.6 a t m abs. for 21 h was used. The distribution of some types of collagenous and non-collagenous proteins in Ascaris cuticle and body wall Table I I summarizes our results on the actual amounts of collagenous proteins in Ascaris cuticle and body wall. As the cuticle contains approx. 30 % of dry substance and the hydroxyproline to collagen conversion factor is 25.0 (see ref. 9), there is approx. 21% of collagen in the cuticle dry substance, represented almost entirely by insoluble collagen. In highly purified insoluble cuticle collagen the ploline/hydroxyprohne molar ratio is 14.2 i 0.38. In the body wall the proline/hydroxyproline molar ratio for purified insoluble collagen is 1.89. There is a negligible amount of collagen extractable into I M NaC1 solution. Biochim. Biophys. Acta, 208 (197 o) 467-474

2

I 4~

g~

I SYNTHESIS IN ASCARIS

OF

II

SOME

COLLAGENOUS

atm abs. atm abs. + 2% CO 2 + 28% N 2at + 2% CO 2 at i atmabs. + 2% CO 2 at 1.6atmabs.

AND

NON-COLLAGENOUS

latmabs.

PROTEINS

__

- -

IN

ASCARIS

O.81 j_ O.IO

I n c u b a t i o n time: o h

itmoles A T P / g wet wt.

CUTICLE

0.56 O.61 __ 0.65 0.62

7 h

AND

BODY

0.84 0.59 0.6 7 0.56 0.43

21 h

"WALL

0.72 --0.24 0.33

24 h -0.28 0.6 5 ---

27 h

.--

0.76 ---

44 h

Insoluble collagen Proline Hydroxyproline 0.45 M NaC1 e x t r a c t a b l e c o l l a g e n Hydroxyproline i M NaC1 e x t r a c t a b l e c o l l a g e n Hydroxyproline Non-collagenous proteins Proline

Parameter studied

1.8 5 ± 0 . 2 2 o.98 + o.13 -o.oi9 17.2 ± 3.2

o . 1 3 -t- 0 . 0 2 o.24 25. 4 ± 4.1

B o d y wall

295 ± 23 2o.1 4- 2.1

Cuticle

t~moles/g wet wt.

T h e d a t a a r e b a s e d o n t h e a n a l y s e s of t w e l v e A s c a r i s of b o t h s e x e s , a v e r a g e b o d y w e i g h t 5.5 g a n d a n a v e r a g e l e n g t h of 26 c m . T h e w e t w e i g h t of t h e c u t i c l e r e p r e s e n t s 7 % a n d t h a t of t h e b o d y w a l l 32 o'o of t o t a l b o d y w e i g h t . V a r i a b i l i t y of t h e r e s u l t s is g i v e n b y S . D .

CONTENT

TABLE

Air at i Air at 3 7o % 0 2 98 % 0 2 98 % 0 2

Experimental conditions

A s c a r i s w e r e i n c u b a t e d i n t h e m o d i f i e d m e d i u m of ELLISON 4 a t 37 ° u n d e r g i v e n e x p e r i m e n t a l c o n d i t i o n s . A T P w a s d e t e r m i n e d b y B o e h r i n g e r s t a n d a r d u l t r a v i o l e t a s s a y i n t h e p u l v e r i z e d w h o l e A s c a r i s . E a c h v a l u e is t h e a v e r a g e of a t l e a s t t w o i n d e p e n d e n t d e t e r m i n a t i o n s . T h e v a r i a b i l i t y is g i v e n b y S . D .

THE EFFECT OF 0 2 CONCENTRATION ON A T P

TABLE

m

4~ "-4 O

471

PROLINE HYDROXYLATION IN ASCARIS

The effect of high 02 concentration on the synthesis of hydroxyproline and non-eollagenous proteins in Ascaris cuticle and body wall The high 02 concentration stimulated the formation of hydroxyproline in the cuticle collagen and had no effect on hydroxyproline synthesis in the body wall collagen (Table III). At 02 concentrations of 7° %, the radioactivity of hydroxyproline in the cuticle was more than double that found at N 2 atmosphere. No consistent effect of atmosphere changes on radioactive proline incorporation into non-collagenous protein was noticed. The effect of high 02 concentration on the hydroxylation of soluble and insoluble collagen in the Ascaris cuticle and body wall The proline/hydroxyproline molar ratio as an index of purity approached the value 13.8 in insoluble collagen fraction isolated from the cuticle. The ratio of the neutral salt-soluble fraction ranged between 27.7 and 55.4, indicating varying degrees of contamination by non-collagenous proteins. The most useful index of the hydroxylation was the hydroxy I14C]proline/[14C~proline ratio (last column of Table IV). As can be seen, the incubation of Ascaris in 02 increases the hydroxylation in cuticle neutral salt-soluble collagen 2.8 times (accolding to the specific activity of hydroxyproline) or 2.9 times (proline/hydroxyproline ratio). The effect of increased O3 on the hydroxylation of body wall collagen was different than that observed in the cuticle. In 02 atmosphere the hydroxylation of the body wall collagen was reduced by approx. 30 % when compared to the value obtained with N 2 (Table IV). Effect of varying 02 concentrations on in vivo hydroxylation of collagen in the cuticle and muscle of Ascaris The hydroxylation of collagen samples with a high purity degree given by proline/hydroxyproline ratio (see Table V) is presented as the ratio of specific activities of collagenous hydroxyC14Clproline/E14Clproline. TABLE III EFFECT OF 02 ON SYNTHESIS OF COLLAGENOUS HYDROXYPROLINE AND NON-COLLAGENOUS PROLINE IN ASCARIS CUTICLE AND BODY WALL Adult Ascaris, injected w i t h Ex4C~proline, were incubated for 24 h in the m e d i u m of ELLISONl at 37 ° in given atmospheres. Total collagens were extracted into 0. 3 M h o t trichloroacetic acid f r o m cuticle and b o d y wall and the specific activity of h y d r o x y p r o l i n e was determined. Trichloroacetic acid precipitate represents non-collagenous proline radioactivity, which is given as per m g of protein.

Atmosphere

N2 70% 02

Cuticle

Body wall

Non-collagenous ?4C~proline (disint./min per mg protein)

Collagenous Non-collagenous E14G~hydroxyproline L14C~proline (disint.]min (disint./min per izmole) per mg protein)

Collagenous E14C~hydroxyproline (disint.]min per l~mole)

6540 8790 614o 6800

558 658 13o6 142o

3031 3089 3o37 335 °

4508 -4115 4615

Biochim. Biophys. Acta, 208 (197 o) 467-474

472

M. CHVAPIL, E. EHRLICH

TABLE IV EFFECT OF 02 ON

in vivo SYNTHESIS OF COLLAGENOUS HYDROXYPROLINE IN

ASCARIS

Ascaris were i n j e c t e d w i t h [14C]proline a n d i n c u b a t e d for 21 h in N 2 or in a i r a t 1.6 a t m abs. T h e i n c u b a t i o n m e d i u m w a s a e r a t e d w i t h t h e gas a t t h e s t a r t of i n c u b a t i o n a n d a g a i n a f t e r 12 h. F r o m t h e cuticle h o m o g e n a t e t w o t y p e s of collagens were i s o l a t e d f r o m w h i c h i ns ol ubl e collagen r e p r e s e n t s a h i g h l y purified sample. I n s o l u b l e collagen w a s i s o l a t e d f r o m t h e m u s c l e only.

Atmosphere

Proline/Hy- E14C]Hydroxy- El~C]Proline E14C]Hydroxydroxyproline proline (counts/rainproline/[14C](iJmoles) (counts/rain per itmole) proline specific per itmole) activity

Cuticle 0.45 M NaC1 e x t r a c t a b l e collagens

N~

27. 7

02 I n s o l u b l e co llagen N2 02

55.4 13.8 13.9

6 5o3 2.8 × 17 914

61°3 58o7

ioi ioo

244 146

I oo 3 670

6o2o 5200

i .06 3.o8

2. 9 x

o.4o8 1 . 7 × o.685

Muscle I n s o l u b l e collagen No 02

2.36 2.48

o.17 o.13

TABLE V EFFECT OF VARYING 02 CONCENTRATIONS ON THE HYDROXYLATION OF COLLAGENOUS PROLINE IN ASCARIS CUTICLE AND MUSCLE Two w o r m s were i n c u b a t e d for 24 h in screw-cap b o t t l e s in 25 ° m l m e d i u m , w h i c h were e q u i l i b r a t e d e v e r y 8 h w i t h g i v e n a t m o s p h e r e (15 m i n b u b b l i n g a t flow r a t e 700 inl/min). O t h e r e x p e r i m e n t a l c o n d i t i o n s were t h e s a m e as g i v e n in T a b l e IV.

Atmosphere

(vol. %)

N 2

5 % O'2 20 ° o 02 7 O oo' 02

Cuticle

Muscle

-Proline/hydrozyproline (/*moles)

Hydroxyproline/proline (specific activity)

Proline/hydroxyproline (t,moles)

Hydroxyproline/proline (specific activity)

II.8 11"7 10.6 11.9

0.77 1'45 0.97 4"65

1-93 1-77 1.98 L75

0.204 0.292 0.492 °-I64

It is apparent that the maximal hydroxylation of collagenous proline in the muscle has been reached at 2o % 02 atmosphere whereas at 7o % 02 hydroxylation was no better than at N 2 atmosphere. More than a 4-fold increase of hydroxylation was found in cuticle collagen at 7 ° % 02. No substantial effect was observed in cuticle samples with N2, 5 and 2o % 02 concentrations. DISCUSSION

The principal object of this study was to investigate the influence of environmental 02 concentration at normal or increased atmospheric pressure on the Ascaris cuticle and body wall collagen hydroxyproline formation. Our hypothesis postulated Biochim, Biophys. Acla,

208 (197 o) 467 474

PROLINE HYDROXYLATION IN ASCARIS

473

a difference in the availability of 02 in tissue structures that would be responsible for the synthesis of an underhydroxylated collagen in the cuticle and a more completely hydroxylated collagen in the body wall. To test the hypothesis we decided to incubate Ascaris at higher 02 concentrations (7° %) for at least 24 h to increase the Po2 in the tissues. The data from the literature suggest that elevated 02 concentration in the environment m a y be toxic to the Ascaris. Our experiments agree with those of BUEDINGa° who indicated that Ascaris will take up O 2 and will survive in environments with increased 02. The survival time, as determined by the ATP synthesizing capacity at various 02 concentrations and by measuring the contraction of muscles afte~ longitudinal dissection, indicates that even a mixture of 98 % 02 + 2 % CO 2 is not lethal since most of the parasites survived for 24 h, provided that the p H of the incubation medium was kept constant; therefore the presence of antibiotics in the medium is essential. Asearis incubated in a medium bubbled thoroughly for at least 15 min with N 2 and further incubated in a desiccator under N 2 atmospheie were used as a control sample. Using this procedure only traces of 02 were left in the medium (controlled by a Beckman oxygenmeter microelectrode system). Therefore, we avoided the incubation conditions used by FUJIMOTO 11 as we had found that removing 02 by evacuation with a water aspirator for 5 min was inefficient. Our study shows the diffeiing effects of high 02 on the hydroxyproline synthesis by cells that produce cuticle collagen and by cells in the body wall that produce muscle collagen. Whereas in the cuticle the hydroxyproline content increased up to 3 times in the neutral salt-soluble collagen, in the body wall the extent of hydroxylation slightly decreased or remained the same. The experiment with varying 02 concentrations on the hydroxylation of collagenous proline in Ascaris cuticle and muscle showed that hypodermis and muscle require different 02 concentrations to secure m a x i m u m hydroxylation. FUJIMO*O11 also had found a higher hydroxyproline formation in cuticle collagen of Ascaris using the "standard" O2-containing medium despite his different method of inducing the required gas atmosphere. In the same study he had described an increase of hydroxyE14C]proline synthesis in the body wall collagen, while in our experiments we found rather an inhibition. However, this might be due to the higher 02 used in our study, which shows that maximal hydroxylation of muscle collagen was reached at 20 % 02 and at 7° % was rather lower than at N 2. In agreement with this result, FUJIMOTO AND PROCKOPTM found that protocollagen proline hydroxylase isolated from the Ascaris body wall differed from the chick embryo enzyme in that it was partially inhibited by incubation in atmospheric air, and the optimal 02 concentration was about 1%. Two explanations are offered for the differences in susceptibility to 02 tension : (I) The cells of the hypodermis contain a different protocollagen hydroxylase than do the cells of the muscle layer and (2) the amount of 02 available for the protocollagen hydroxylase activity m a y be different in the two structures. It is not possible to present direct evidence bearing on these two possibilities, but one might speculate that the difference in response of the two layers to increased 02 tension m a y be related to the findings of DAVENPORT13,14, who showed 3 times more oxyhemoglobin in Ascaris hypodermis than in the muscle layer. Our data on the distribution of collagen in Ascaris cuticle and body wall show Biochim. Biophys. Acta, 208 (197 o) 467-474

474

M. CHVAPIL, E. EHRLICH

a rather low content of collagen in the cuticle membrane. The proline/hydroxyproline ratio found in the insoluble cuticle collagen agrees with most of the literature data 1-3. The value of the proline/hydroxyproline molar ratio for insoluble body collagen amounts to 1.89, which would agree with the results of FUJIMOTO AND ADAMS3 but is rather high according to a recent paper of FUJIMOTO15. This author used a more drastic purification procedure and arrived at the conclusion that collagen from muscle layers of Ascaris contains more hydroxyproline than proline. In his review on the biochemistry of Ascaris, tTAIRBAIRN16 stresses that the respiration of this parasite in pure 02 is 3 times as fast as in air. Despite this fact, we did not find a consistent change in the incorporation of proline into non-collagenous proteins, either in the cuticle or in the body wall, thus tending to eliminate a nonspecific effect of 02 on protein synthesis of the Ascaris. ACKNOWLEDGMENTS

This study was supported in part by a Max Planck Geselschaft Grant and by U.S. Public Health Service Grand HE 11035 and Training Grant HE 05463 from the National Heart and Lung Institute, and performed in part at the Max Planck Institut ftir Eiweiss und Lederforschung, Munich, Germany. The authors acknowledge with gratitude the critical suggestions of Dr. Robert J. Boucek, and the excellent technical assistance of Mrs. Jifina Magkov~ and Mrs. Irena ~t6rbov~. REFERENCES I 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16

J. J o s s E AND W. F. HARRINGTON, dr. Mol. Biol., 9 (1964) 269. M. R. WATSON AND N. R. SILVESTER, Biochem. dr., 71 (1959) 578. D. FUJIMOTO AND F ADAMS, Biochem. Biophys. Res. Commun., 17 (1964) 437. T. ELLISON, P h . D . Thesis, University of Wisconsin, 1959, Univ. Microfilm LC Card, No. Mic. 59-3184, 1959, p. I4O. S. M. FITCH, IVi. L. R. HARKNESS AND R. D. HARKNESS, Nature, 176 (1955) 163. W. TROLL AND J. LINDSLEY, J. Biol. Chem., 215 (1955) 655. H. STEGEMANN, J. Physiol. Chem., 371 (1958) 41. A. P. HOBSON, Parasitology, 38 (1948) 183. D. S. JACKSON AND E. G. CLEARY, in D. GLICK, Methods of Biochemical Analysis, Vol. 15, Academic Press, New York, 1967, p. 25. E. BUEDING, Physiol. Rev., 29 (1949) I95. D. FUJIMOTO, Biochim. Biophys. Acta, 14o (1967) 148. D. FUJIMOTO AND D. J. PROCKOP, J. Biol. Chem., 244 (1969) 2o5. H. E. DAVENPORT, Proc. Roy. Soc. London, Set. B, 136 (1969) 255. H. E. DAVENPORT, Proc. Roy. Soe. London, Ser. B, 136 (1969) 281. D. FUJIMOTO, Biochim. Biophys. Acta, 168 (1968) 537. D. FAIRBAIRN, Exptl. Parasitol., 6 (1957) 491.

Biochim. Biophys. Acta, 2o8 (197 o) 467-474