Biochimica et Biophysica Acta, 304 (1973) 457-465 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

BBA 27119

INFLUENCE OF ZINC DEFICIENCY ON SYNTHESIS AND CROSS- LINKING OF RAT SKIN COLLAGEN

P. E. McCLAIN, E. R. WILEY, G. R. BEECHER, W. L. ANTHONY and J. M. HSU

Nutrition Institute, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Md. 20705 (U.S.A.) and Biochemistry Research Laboratory, Veterans Administration Hospital, Baltimore, Md. 21218 (U.S.A.)

(Received October 4th, 1972)

SUMMARY

(1) The yield of salt-soluble collagen was significantly reduced in zinc-deficient rats, while the acid-soluble fraction was increased by approximately 20 ~.

(2) The incorporation of [2-1#C]glycine into the ~1 and ct 2 chains and of r-[U-14C]proline into the salt-soluble collagen fractions was reduced 30--50 ~ in the zinc-deficient animals.

(3) The incorporation of [Me-aH]thymidine into skin DNA was 60--80 ~o lower than that from zinc-supplemented animals.

(4) Muscle collagen synthesis per mg polyribosomal RNA was approximately the same in both groups. The polysome yield and consequently total muscle collagen synthetic activity per 100 g muscle was, however, 30 ~ lower in the animals on a zinc-deficient diet.

(5) The content of fl-dimers in the salt-soluble collagen from zinc-deficient skins was 1/3 higher than that from the control. No difference in fl-subunits from the acid-soluble fraction was apparent.

(6) The aldehyde content of the salt-soluble collagen was markedly higher and that from insoluble collagen lower in the zinc-deficient group.

INTRODUCTION

The marked abnormalities in tissue growth and repair processes produced by zinc deficiency in man and animals have long been recognized 1. It is becoming increasingly evident that these deficiency symptoms are produced, at least in part, by the effect zinc exerts on protein and amino acid utilization and metabolism. The results of Nielsen e t al. 2,3 for example, indicate that zinc-deficiency symptoms in chicks can be partially alleviated by histidine or cysteine supplementation. The essen- tiality of zinc in the regulation of reduced glutatb.ione 4, and the increased oxidation of many amino acids in zinc-deficient rats have also been reported 5,6.

Many workers have concluded that zinc plays a fundamental role in protein biosynthetic activity 7- 9. The observations that zinc is required for protein synthesis

458 P.E. McCLAIN et al.

in Rhizopus nioricans 1° and Euglena 9racilus 11 and is also necessary for hepatic DNA synthesis and amino acid utilization in the rat ~2" ~3 have lent strong support to this conclusion.

Recent evidence suggests that zinc may also be essential for wound healing t 3- ~ 6 An acceleration of the rate of wound repair reportedly follows the administration of zinc in both man and animals. Since collagen is the most important protein to be synthesized in the process of connective-tissue repair and is primarily responsible for gains in wound tensile strength, much interest is currently being focused on the effect of zinc in collagen metabolism. A number of workers have reported that maintaining animals on a zinc-deficient diet results in an abnormal condition in skin and hair 1, ~ 7, ~8 Further work has revealed that zinc deficiency drastically reduces the quantity of [l-t4C]glycine, L-[U-14C]proline and L-[U-t4C]lysine incorporation into rat skin collagen ~9. These studies also revealed that there were no marked changes in the uptake of these amino acids into liver, kidney, testes, or muscle protein, suggesting that zinc may be more important in the metabolism of skin collagen than in other tissue proteins.

The work herein described was initiated to further elucidate the rote of zinc in collagen metabolism. A preliminary report of this research has been presented z°.

MATERIALS AND METHODS

Selection and treatment o f samples Zinc-deficient and pair-fed zinc-supplemented animals were fed and housed as

previously described 21. Skin samples were removed immediately after sacrifice, shaved free of hair and frozen on dry ice. The skin samples were subsequently freeze-dried and passed through a Wiley mill with powdered dry ice.

The neutral-salt and acid-soluble collagen from the powdered skin samples was extracted and purified as previously outlined 2z. Aliquots were removed from the two soluble fractions and the hydroxyproline content determined by the method of Woessner z3. The insoluble residues obtained from the above extractions were further purified according to the method of Jackson and Cleary z4 to a final hydroxyproline content of 13.6 ~ .

Subunit isolation and composition The purified soluble collagen fractions were subjected to acrylamide disc gel

electrophoresis according to the method of Tanzer et al. zS, and the component composition determined by densitometer tracing of the band pattern on a Canalco Model E microdensitometer. The ~ and ~2 chains were isolated by chromatography on a 1.5 cm × 7.5 cm column of carboxymethyl cellulose (Whatman CM-52) at 42 °C as outlined previously 26.

Aldehyde determination The aldehyde content of the collagen fractions was determined by the colori-

metric assay of Paz et al . 27 a s modified by Chou et al. 28.

Incorporation studies Extraction and determination of skin DNA was accomplished using the

INFLUENCE OF ZINC DEFICIENCY ON COLLAGEN 459

method of Schmidt and Thannhauser 29. In vivo incorporation studies utilizing labeled amino acids or thymidine were performed as previously described 19. Components for in vitro protein biosynthesis were isolated fl"om skeletal muscle according to the procedures of Bjercke, R. J. and Beecher, G. R. (personal communication). In vitro muscle collagen biosynthesis activity was assayed by the methods of Ionasescu et al.3 o.

Amino acid analysis

Collagen samples were hydrolyzed at 110 °C for 24 h as previously outlined al. Aliquots of about 100/zg of protein were analyzed in a Technicon TSM amino acid analyzer (Tarrytown, N.Y.).

RESULTS

The yield of salt-soluble collagen from the skins of zinc-supplemented and zinc-deficient rats is shown in Table I. These data show a 20 % decrease in the salt- soluble fraction from the zinc-deficient animals, indicating a possible reduction in protein synthesis. In support of this conclusion, the data shown in Table I1 reveal

TABLE I

YIELD OF SALT- AND ACID-SOLUBLE COLLAGEN*

Diet mg collagen per g skin

Salt-soluble Acid-soluble

Zinc-supplemented Mean 43.4 49.1 S.E. 0.8 0.9

Zinc-deficient Mean 34.9 58.2 S.E. 1.2 1.4

* Mean and standard error, twelve samples per treatment.

TABLE II

INCORPORATION OF [2-14C]GLYCINE INTO TI-[E oq AND 0~2 CHAINS AND L-[U-14C]- PROLINE INTO SALT-SOLUBLE RAT SKIN COLLAGEN*

Diet [2-' 4C] Glycine dpm per mg collagen

OC 1 ~t 2

L- [U- 14C]Prolin e dpm per mg salt-soluble collagen

Zinc-supplemented Mean 4254 3785 340 S,E. 124 80 18

Zinc-deficient Mean 2243 2641 163 S.E. 98 141 16

* Mean and standard error, six samples per treatment.

460 P . E . M c C L A I N et al,

a reduction in the incorporation of [2-14C]glycine into the 0~ 1 and ~2 chains of salt- soluble rat skin collagen. The specific activity in the ~1 and ~2 chains from the zinc- deficient rats was 49 and 30 % less, respectively, than that from the zinc-supplemented animals. The specific activity of the salt-soluble collagen from zinc-supplemented and zinc-deficient animals injected with L-[U-agC]proline 24 h prior to sacrifice is also shown in Table II. Here again a reduction in incorporation of more than 50 "~i was observed in the collagen from zinc-deficient animals.

The incorporation of [Me-3H]thymidine into rat skin DNA was also reduced 56-78 % in the zinc-deficient animals (Table IIl). The total quantity of skin DNA was 4-8 % lower in zinc-deficient animals; however, these differences were not statisti- cally significant.

T A B L E III

I N C O R P O R A T I O N O F [Me-aH]TI - [YMIDINE I N T O R A T SKIN D N A *

Diet Hours after DNA concentration Specific activity injection ttg DNA-P/g dpm/lO0 #g DNA

ZnS I 151 ± 1 0 6 9 6 ± 140 Z n D I 139±26 150± 75 ZnS 2 171:i 5 5 2 7 ! 47 Z n D 2 1642 9 2 3 0 2 58

* Mean :~ s tandard error, six samples per t rea tment .

All of the above studies strongly link zinc to the biosynthetic activity of rat skin collagen. In an attempt to pinpoint the site of zinc influence in the biosynthetic process, the in vitro synthesis of rat muscle collagen was investigated. Table IV shows the results of these studies. It is evident that muscle collagen synthesis, which reflects the incorporation of L-[U-14C]leucine into protein per mg of polyribosomal RNA was approximately the same in both the zinc-supplemented and zinc-deficient animals. The polysome yield, which reflects the/~g of polyribosomal RNA/mg muscle was, however, approximately 32 % lower in the zinc-deficient animals. Consequently, the total muscle collagen synthetic activity per 100 g muscle was 30 % lower than that from the zinc-supplemented animals.

T A B L E IV

IN VITRO M U S C L E C O L L A G E N S YNT HE S IS

Diet Muscle collagen Polysome Total muscle collagen synthesis* yield** synthetic activity***

Zinc-supplemented 24.1 41.4 99.8 Zinc-deficient 24.8 28.3 70. l

* pmoles L- [U- '4C] leuc ine incorpora ted into p ro te in /mg polyr ibosomal R N A per 15 min . ** /tg po lyr ibosomal R N A / g muscle.

*** pmoles L-[U-lgC]leucine incorpora ted into protein per 100 g muscle per 15 rain.

INFLUENCE OF ZINC DEFICIENCY ON COLLAGEN 461

O.E

0 . . ~

t , t

z 0~ m n~

0

0 .~

0.1

o,I

L

. . . . Zn-

- - Z n +

/ / / ' , / ' , // ~/"~,

. . . . . . . . . , d v - . - ' ,¢,=.=.~_.=.-

SCAN

Fig. 1. Densitometer tracing of disc gel patterns obtained from salt-soluble collagen.

Densitometric tracings of the disc gel patterns from zinc-supplemented and zinc-deficient salt-soluble collagen are shown in Fig. 1. The fl-components from the zinc-deficient animals are obviously increased in comparison to that from the zinc- supplemented animals. Concomitantly, the a-monomers are decreased. The greater than 30 % increase in fl-dimers suggests a greater degree of intramolecular cross- linking in the salt-soluble fractions from zinc-deficient skins. In confirmation of these findings, the aldehyde content of the salt-soluble collagen from the zinc-deficient animals was almost twice as high as that from the zinc-supplemented animals (Fig. 2). The aldehyde content of the insoluble skin collagen was, however, almost 47 % lower in the zinc-deficient animals than in the zinc-supplemented animals (Fig. 2), suggest- ing the possible participation of zinc in the intermolecular cross-liking process.

2.2

1.8

~E 1.4

0 0

:Zl- LO

0.6

0.2

S A LT - - SOLUB LE

ALDEHYDE CONTENT

T I

J

Zn

INSOLUBLE

Fig. 2. Aldehyde content of salt-soluble and insoluble skin collagen. Mean and range of six samples per treatment.

462 P.E. McCLAIN et al.

0.4

O.]

0.2

OA

E c

o

o (~ 0 4 <~

0 3

0.2

0.1

J

CLI

B

i i i

B,I 312 (~2

\

,~o ,~o ~o EFFLUENT VOLUME

Fig. 3. Carboxymethyl cellulose chromatogram of heat-denatured acid-soluble collagen. A, zinc- deficient; B, zinc-supplemented.

From the data shown in Table I, it is also apparent that the acid-soluble collagen pool from zinc-deficient animals was increased by nearly 20 ~. The ~/fl

ratios of the acid-soluble fractions did not, however, vary markedly between the zinc- supplemented and zinc-deficient samples (Fig. 3). The amino acid composition of the ~1 and ~2 chains of skin collagen from zinc-supplemented and zinc-deficient animals is shown in Table V. No marked variations in s-chain amino acid composition are apparent in either experimental group, suggesting that zinc deficiency had no effect on the primary-secondary structure of the collagen molecule.

DISCUSSION

The collagen extracted in neutral salt solutions is considered to be the most recently synthesized collagen a2,33. The decreased yield of this fraction in skins from zinc-deficient animals, although suggesting decreased protein synthesis, does not provide definitive evidence for this conclusion. A decrease in the salt-soluble pool could also be explained in terms of increased collagen catabolism in the zinc-deficient tissues. The decreased incorporation of glycine into the a-chains and proline into the salt-soluble pool does, however, tend to support the conclusion that zinc plays a definitive role in the biosynthetic activity of skin collagen.

The unaltered in vitro polyribosome synthesizing ability in the deficient animals indicates that the translation step is probably proceeding at a normal rate. However, the decreased polyribosome yield and concomitant lowering of total muscle collagen

INFLUENCE OF ZINC DEFICIENCY ON COLLAGEN 463

TABLE V

AM INO ACID COMPOSITION OF ~-CHAINS FROM SALT-SOLUBLE RAT SKIN COL-

LAGEN*

Amino acid Residues per 1000

Zinc-supplemented Zinc-deficient

~1 ~2 ~I ~2

Aspartic 47 46 47 45 Hydroxyproline 94 92 94 90 Threonine 20 24 20 24 Serine 40 45 40 43 Glutamic 75 75 75 73 Proline 119 105 118 107 Glycine 329 335 329 332 Alanine 110 106 114 108 Valine 20 20 20 20 Methionine 7 4 7 4 lsoleucine 14 10 11 10 Leucine 23 26 20 27 Tyrosine 3 3 3 3 Phenylalanine 12 13 15 13 Hydroxylysine 3.5 9.1 3.3 8.2 Lysine 32.5 25.8 32.6 26.7 Histidine 2 10 2 10 Arginine 49 50 49 53

* Mean of three samples per treatment.

synthetic activity points to an alteration in RNA synthesis induced by zinc deficiency. The reduced incorporation of [Me-3H]thymidine into skin DNA further suggests that the zinc-induced defect may also reside in impaired DNA replication.

The results of this study do not rule out the possibility that zinc deficiency is adversely affecting other factors which could alter collagen synthesis, such as an inhibition of the prolyl and lysyl hydroxylases or glycosylation processes which would prevent release of newly synthesized collagen from the fibroblast 34. The present results do, however, tend to lend strong support to previous findings 4'5'9' 19 which suggest that zinc deficiency results in a marked impairment of DNA-RNA synthesis and replication, leading to a reduction in ability of the connective tissue to synthesize collagen.

The results of this investigation also suggest that zinc may play a fundamental role in the collagen cross-linking process. Certainly the increased content of fl-compo- nents and increased aldehyde content of the salt-soluble collagen pool indicates that the formation of covalent intramolecular cross-links is enhanced in the skin collagen from zinc-deficient rats. The reason for this increase in intramolecular cross-linking is not clear from the results of the present study. It is known, however, that the tissues of zinc-deficient animals have a high content of copper (Hsu, J. M., personal commu- nication). Copper is known to be an essential cofactor for amine oxidase, the enzyme which oxidatively deaminates lysine to allysine, the precursor of the collagen intra- molecular cross-link 2s.

464 P.E. McCLAIN et al.

The increased yield of acid-soluble collagen further suggests that zinc deficiency may also be altering intermolecular covalent cross-linking. In this case, however, zinc deficiency apparently has resulted in an inhibition of the cross-linking mechanism. The collagen extracted in dilute acid solutions is thought to be biologically older than that from the salt-soluble pool, and is derived from the aggregated collagen fiber. Consequently, an increase in this fraction indicates a decrease in the number of stable intermolecular cross-links, probably of the aldimine type 34. The reduced aldehyde content in the insoluble skin collagen may also reflect a lowered potential for cross- link formation 34. The dichotomy of increased intramolecular cross-linking in the salt-soluble pool, accompanied by an apparent decrease in intermolecular cross- linking as reflected by the increased yield of acid-soluble collagen is not altogether clear. We have, however, observed similar results in studies of collagen isolated from the intramuscular connective tissues of striated muscle 31.

In any case, the reduced collagen synthesis and apparent alterations in collagen cross-linking observed in zinc-deficient rat skin could explain the enhanced wound healing attributed to zinc administration in vi tro ~4" 16 as well as the wound healing problems observed in zinc-deficient humans 35.

REFERENCES

I Todd, W. R., Elvejhem, C. A. and Hart, H. B. (1934) Am. J. Physiol. 107, 146-156 2 Nielsen, F. H., Sunde, M. L. and Hoekstra, W. G. (1966) J. Nutr. 89, 35-42 3 Nielsen, F. H., Sunde, M. L. and Hoekstra, W. G. (1967) Proc. Soc. Exp. Biol. Med. 124, 1106-

1110 4 Hsu, J. M., Anthony, W. L. and Buchanan, P. J. (1968)Proc. Soc. Exp. Biol. Med. 127, 1048--1051 5 l-[su, J. M., Anthony, W. L. and Buchanan, P. J. (1969) J. Nutr. 97, 279-285 6 Theuer, R. C. and Hoekstra, W. G. (1966) J. Nutr. 89, 448454 7 Fujioka, M. and Lieberman, 1. (1964) J. BioL Chem. 239, 1164-1167 8 Williams, R. B., Mills, C. F., Quarterman, J. and Dalgarno, A. C. (1965) Biochem. J. 95, 29 p 9 Sandstead, H. H. and Rinaldi, R. A. 0969) J. Cell. Physiol. 73, 81-83

l0 Wegner, W. S. and Romano, A. H. (1963) Science 142, 1669 11 Wacker, W. E. C. (1962) Biochemistry I, 859-865 12 Vallee, B. L. (1959) Physiol. Rev. 39, 443-490 13 Strain, W. H., Pories, W. J. and I-Iinshaw, J. R. (1960) Sur9. Forum 11,291-292 14 Pories, W. J., Henzel, J. 1-[., Rob, C. G. and Strain, W. H. (1967) Ann. Sur9. 165, 432436 15 Cohen, C. (1968) Br. Med. J. 2, 561 16 Pories, W. J., I-[enzel, J. H., Rob, C. G. and Strain, W. H. (1967) Lancet 1, 121-124 17 Ott, E. A., Smith, W. H., Stob, M. and Beesen, W. M. (1964) J. Nutr. 82, 41-50 18 Macapinlac, M. P., Barney, G. H., Pearson, W. N. and Darby, W. J. (1967) J. Nutr. 93,499-510 19 I-[su, J. M., Anthony, W. L. and Davis, R. L. (1971) Fed. Proc. 30, 643 20 McClain, P. E., Wiley, E. R., Anthony, W. L. and 1-[su, J. M. (1972) Fed. Proc. 31, 669 21 I-[su, J. M., Anthony, W. L. and Buchanan, P. J. (1969) J. Nutr. 99, 425-432 22 McClain, P. E. and Wiley, E. R. (1972) J. Biol. Chem. 247, 692-697 23 Woessner, J. F. (1961) Arch. Biochem. Biophys. 93, 440~47 24 Jackson, D. S. and Cleary, E. G. (1967) in Methods of Biochemical Analysis (Glick, D., ed.),

Vol. 15, pp. 25-76, Interscience, New York 25 Tanzer, M. L., Monroe, D. and Gross, J. (1966) Biochemistry 5, 1919-1926 26 Morris, S. C. and McClain, P. E. (1972) Biochem. Biophys. Res. Commun. 47, 27-34 27 Paz, M. A., Blumenfeld, O. O., Rojkind, M., I-[ensen, E., Furfine, C. and Gallop, P. M. 11965~

Arch. Biochem. Biophys. 109, 548-559 28 Chou, W. S., Savage, J. E. and O'Dell, B. L. (1969) J. Biol. Chem. 244, 5785-5789 29 Schmidt, G. and Thannhauser, S. J. (1945) J. Biol. Chem. 161, 83-89 30 lonasescu, V., Zellweger, H. Z., Filer, L. J. and Conway, T. W. (1970) Arch. Neurol. 23, 128-136

INFLUENCE OF ZINC DEFICIENCY ON COLLAGEN 465

3t McClain, P. E., Creed, G. J., Wiley, E. R. and Gerrits, R. J. (1970) Biochim. Biophys. Acta 221, 349-356

32 Lowther, D. A. (1963) in Int. Rev. Connective Tissue Res. (Hall, D. A., ed.), Vol. 1, pp. 63-119, Academic Press, New York

33 Jackson, D. S. and Bentley, J. P. (1960) J. Biophys. Biochem. Cytol. 7, 37-42 34 Traub, W. and Piez, K. A. (1971) in Advances in Protein Chemistry (Anfinsen, C. B., Edsall,

J. T. and Richards, F. M., eds), Vol. 25, pp. 243-352, Academic Press, New York 35 Henzel, J. H., DeWeese, M. S. and Lichti, E. L. (1970) Arch. Surg. 100, 349-357