Alterations of post-translational modifications of procollagen by SV40-transformed human fibroblasts

Volume 85, number 1

ALTERATIONS

FEBS LETTERS

OF POST-TRANSLATIONAL DY SV40-TI~,NSFORMED

January 1978

MODIF~CAT_rO,NS OF PROCOLLAGEN HUMAN F|BROBLASTS

Nirmala S U N D A R R A J * and R o b e r t L. C H U R C H + Departments o f Ophthalmology* and A n a t o m y and Cell ~iology ÷, University o f tYttsburgh School o f l~Iedicine, Pittsburgh, PA ~5213, USA

Received 13 October 1977

[. [ n t r o d u c t i o n It is n o w well k n o w n that collagen is initially synthesized as a precursor called proeoliagen, which t h e n undergoes a variety o f post-translational modifications to yield collagen. These modifications include h y d r o x y l a t i o n o f proline and lysine residues, glycosylation o f certain h y d r o x y l y s i n e residues, and cleavage o f the c a r b o x y - and a m i n o - t e r m i n a l p e p t i d e extensions f r o m the procollagen molecule. Although the procoiiagen intermediates are shortqived in vivo, their conversion into collagen is relatively slow in cells growing in culture [ 1 ] . N o r m a l h u m a n dermal fibroblasts growhng in culture are k n o w n to secrete procollagen into the m e d i u m [ 1 - 6 ] . These cells also secrete procoHagen peptidase(s) which cleave the terminal p e p t i d e extensions o f procollagen to f o r m collagen [ 7 ] . Viral t r a n s f o r m a t i o n o f ce~ls in culture is k n o w n to result in changes in g r o w t h patterns, specific activity o f e n z y m e s , cell surface, and nuclear and m e m b r a n e associated antigens [ 8 ] . This c o m m u nication gives evidence that viral t r a n s f o r m a t i o n also affects the p o s t s y n t h e t i c m o d i f i c a t i o n o f procollagen. Those activities t h a t a p p e a r to be altered b y transf o r m a t i o n include procollagen peptidase activity as well as the e x t e n t o f lysine h y d r o x y l a t i o n and glycosylation in the procollagen m o l e c u l e .

2. Materials a n d m e t h o d s An S V 4 0 - t r a n s f o r m e d h u m a n fibroblast line ( G M 6 3 7 ) and its u n t r a n s f o r m e d n o r m a l diploid fibroblast parent ( G M 3 7 ) were o b t a i n e d f r o m the Inst. t~'lsevier/North-Arolland B~'omedieag Fress

Med. Res. Camden, NJ. The cells were grown as m o n o l a y e r s either in plastic tissue-culture dishes or _oiler b o t t l e s in W a y m o u t h m e d i u m 75211 ( G i b c o ) s u p p l e m e n t e d w i t h 10% fetal-calf serum and 100 txg/ ml ascorbic acid, w i t h o u t antibiotics. When the cells f o r m e d m o n o l a y e r s , the m e d i u m was changed to either serum-free 75211 s u p p l e m e n t e d wffh t 0 0 / a g / ml ascorbic acid and 20 ng/m! growth tripepfide (Ca!biochern) or with D u ! b e c c o ' s m o d i f i e d Eagle's m e d i u m (DME) nfinus serum, plus 1 0 0 / l g / m l ascorbic acid, 20 ng/ml g r o w t h tripeptide and 1 /zCi/ml [3H]proline. ~lae culture m e d i u m was removed fro~a the cells after 2--3 days i n c u b a t i o n and the procollagens and collagens vgere purified t h r o u ~ a DEAEcellulose c h r o m a t o g r a p h y a,i reported [ 9 ] .

3. Results and discussion DEAE-cellutose c h r o m a t o g r a p h y o f p r o c o l l a g e n isolated f r o m the culture m e d i u m o f n o r m a l fibroblasts ( G M 3 7 ) is seen in fig. 1A. Fractions p o o l e d as indicated were further analyzed b y SDS polyacrylam/de gel electrophoresis. Sensitivity o f these fractions to collagenase and pepsin, :;nd their crossreactivity with antisera prepared against purified t y p e I and t y p e IIl h u m a n procollage:as were also tested. Peak 1, which eiuted f r o m the c o l u m n in the b u f f e r wash, was f o u n d to contain collagen molecules compos~zd o f o~1 and a 2 chains a p p r o x . 95 0 0 0 tool. wt. Peak 2 c o n t a i n e d t y p e I procollagen having a chain c o m p o s i t i o n o f [pro o~1([)] 2pro ~2 at a p u r i t y o f m o r e than 95% as judged b y collagenase sensitivity (fig.2A). 47

Votum,~ 85, number 1

FEBS I EIFrERS

January. 1978

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Fig.IA_B_ DEAE-cellu!ose chromatography of partially purified procoUagens obtained from (A) normal fibrobtasts (GM37); ([1) SV,[0-transformed G~I-~7 (G_=%1637),grown as described in the t¢:xt. Procotlagens were purified from serum-free culture media from tKe cell~ as described [9]. This material was dissolved in 0.05 IH Tris-HCl (pH 7.4) containing 2 M urea arid G.3 M NaC1 (limiting buffer) and dialyzed against 0.05 M Tris-HC1 (pH 7.4) coutaining 2 M urea (starting buffer) and applied to a column (1.5 × 20 cm) cf DEAE-ceUuiose equilibrated in the starting b-_'fCer.The column was developed with a linear gradient of limiting buffer (eonductivit:.- 17 milli MHO) using a Gilson Mixograd automatic gradient former. The fractions in individual peaks were separat,.'ly pooled, dialyzed extensively against distilled water and lyophilized.

Pro a l a n d p r o a 2 c h a i n s m i g r a t e d w i t h a p p r o x . tool. ~vt 120 0 0 0 i n S D S - - p o i y a c ~ , l a m i d e gels. L i m i t e d p e p s i n d i g e s t i o n o f the p e a k 2 p r o c o l l a g e n f r a c t i o n

yielded a l and a 2 chains approx. 95 000 mol. wt having an c,.1:a2 ratio o f 2:1. More than 90% o f the protein ehiting in peak 3 (fig-lAt) were c o l l a g e n o u s a n d reacted b o t h w i t h

type I and type IiI antisera. Although peak 4 had type Ii] procollagen with no type I contamination

a_rnide gels (fig.2C). T b e o~-chahas o b t a i n e d b y l i m i t e d p e p s i n h y d r o l y s i s o f this p r o c o l ! a g e n m i g r a t e d as c h a i n s with tool. w t 1G% larger t h a n collagen a ~ h a i n s f r o m n o r m a l cells (fig.2C). P e a k 2 (fig. 1 B) was f o u n d t o c o n t a i n t y p e [I[ p r o c o l l a g e n h a v i n g p r o 0z c h a i n s w h i c h m i g r a t e d o n SIF)S-gels i n t h e O r e , o n ( m o l . w t 150 009)_ P e p s i n h y d r o l y s i s p r o d u c e d coUagen w i t h ~1 chaio~ w h i c h a p p e a r e d 10% larger i n gels t h a n

norma¢ type IIl collagen (fig.2D). This peak was con-

(fig.2E;), the m a t e r i a l was o n l y 7 5 - - 8 0 % p u r e a n d was c o n t a m i n a t e d w i t h n o n - c o l l a g e n o u s p r o t e i n s . Procollagens p u r i f i e d f r o m SVF'O-transformed cells (GM537) and chromatographed on DEAE-cellulose

t a m i n a t e d w i t h a p p r o x . 20% n o n c o l l a g e n o u s p r o t e i n b u t was free f r o m t y p e I p r o c o U a g e n as j u d g e d b y t h e a b s e n c e o f c~! b a n d s o n p o l y a c r y i a m i d e ~els a n d also the a b s e n c e o f a n y r e a c t i o n w i t h anti-type I a n t i -

(fig. 1E;) eIuted at a different ionicity on DEAE-

serum.

cellulose t h a n p r o c o l l a g e n s f r o m the n o r m a l cells, i n d i c a t i n g t h a t t h e y d i f f e r e d i n their n e t charge. A l s o , n o col!agen p e a k was o b s e r v e d ( t h e small peak w h i c h was e x c l u d e d f r o m the c o l u m n , was f o u n d to be n o n c o l l a g e n o u s ) . Peak I (fig. t B ) c o n t a i n e d t y p e I proc o l l a g e n at m e r e t h a n 95% p u r i t y , h o w e v e r , the p r o c h a i n s m i g r a t e d i n the tool_ w t 150 0 0 0 [pro c~l(l_)] a n d 120 0 0 0 ( p r o a 2 ) regions o n SDS--polyac,_~-l-

48

Amino acid composition o f type [ collagen o f G M 3 7 was very s i m i l a r t o t h a t r e p o r t e d f o r t y p e [

zotlagdn from skin (table 1). The composition o f pepsin-treated type III collagen o f GM37, differed :dightly from that reported for skin Wl?e IlI collagen [ 11 ], probably due to the presence o f m i n o r con~ a m i n a n t s e v e n a f t e r pep-~i_n t r e a t m e n t a n d "~e-precipi-

:ation ofcoUagen. However, the amino acid analyses

V o l u m e 85, n u m b e r i

FEBS L E T T E R S

COHTROL

....

P~ I}SI fd T~Et&~'EO

January 1978

hydro.~y~yshae residues f r o m the t r a n s f o r m e d ceils as c o m p a r e d to n o r m a l (table 2). However, since tire t r a n s f o r m e d cell collagens had twice as m a n y residues o f h y d r o x y l y s l n e as normal, the total percent glyeosylafion o f the hydro×y!ysine residues did not differ significantly (table 2). The a m o u n t o¢ h y d r o x y l a d o n o f prolyl or lysyl residues changes the m~gration o f (~-chaips on SDS p o l y a c r y l a m i d e gels [ 1 2 ] . The increased h y d r o × y l a f i o n observed in the transfomaed cell collagens p r o b a b l y accounts for the a p p a r e n t higher molecula:" weight observed in fig.2C and 2D. T r a n s f o r m e d cells, unlike normal fibroblasts, e x h i b i t e d a l a c k o f p r o z o U a g e n p e p t ~ d a s e a c t i v i t y , as

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judged b y the a c c u m u l a t i o n o f only uncleared proc0Uage~ molecules in the culture media either wtr.h ~:w i t h o u t serum. ProcoUagens isolated f r o m the translooo

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phi~ized samples, 50/~g (solid line), or 50 #g pepsin-treated samples (dashed line) were p r e p a r e d for S D S - - p o l y a e r y t a m i d e gel electrophoresis as described [9] and d e c t r e p h o r e s e d o h 4% polyac_ry~amide ge!s (Bio-Rad) until t h e d y e m a r k e r reached the b o t t o m o f the gel. The gels were stained with

Coomass~e blue, desta~ned and scanned.

indicated a significant d~fferenee it: the degree of

h y d r o × y l a f i o n o f [ys~ne residues b e t w e e n n o r m a l and t r a n s f o r m e d cells. The transformo, d cells d e m o n s t r a t e d a 2-fold increase in hydroxylys~ne residues b o t h in the t y p e i and t y p e _~U,collagens w h e n c o m p a r e d to n o r m a l collagens. Interestingly, there was also a 2-fo!d ~ncrease h~ the t o t a l glycosylat~on o f collagen

Fig.3. S D S - p o l y a c r y l a m i d e gel electrophoresis o f partially purified t y p e I procollagen f r o m S V 4 0 - t r a n s f o r m e d h u m a n fibroblasts (GM637 solid line) and o f GM637 proeollagens treated w i t h a partially purified procotlagen peptidase fract i o n o b t a i n e d f r o m -he m e d i u m o f n o r m a l h u m a n t'ibroblast5

(GM37, dashed fine). The partially purified procollagen peptidase fraction was obtained by the method [7j. The reaction mi×ture containing 90/~1 substrate (5000 c p m p r o collagen f r o m GM637) a n d 10 t~l crud~ etazyme was i n c u b a t e d at 37°C for 2 h. T h e reaction was s t o p p e d b y adding 10 t,! t 0 X SDS sample b u f f e r and heating to 100°C for 3 rain.

Eiectrophoresis of these samples were ,carried out as described for fig.2. Gels were fractionated into I mm sections using a G~son automatic ge~ fractionator. EacEhfraction was collected and e x t r a c t e d ~n 200 t~110% SDS and the radioactivity was determined.

49

V o l u m e 85, n u m b e r 1

FEBS L E T T E R S

Sanuary 1978

Table 1 AmJiao ac{d c o m p o s i t i o n o f procoHat~ens a n d coI!agens (pt.'psin-treated procoHagens) p~z~fied f r o m ¢:ulture m e d i u m o f n o r m a l h u m a n fibroblast ( G ~ 3 7 ) attd S V 4 0 - t r a n s f o r r n e d G M 3 7 ( G M 6 3 7 ) E x p r e s s e d as r e s i d u e s a m i n o a c i d s ] 1 0 0 0 r e s i d u e s Type I pro¢ollagen [ p r o o:l(1)] ~ p r o 0_2

T y p e ,-" collagen [ a l ( I ) ] 2 c~2

GM37

Humana

GM637

GM37

T y p e I l l collagen [~I(1H)] GM637

skin Hydroxyproline A s p a r t i c acid Threoiiine Ser:me Glu tamic- acid Prolme Glycine Alanine Half cystine Valine Met.hionine lsoteucine Leucine Tyrosine fhenylalanine Histidine Hydroxylysine Lysine Tryptophan Arginine

78 53 23 37 75 110 300 ! 12 3.4 27 6.4 16 34 12 20 7.3 8.0 30 1.0 47

83 55 24 40 78 114 299 105 3.3 24 7.2 14 34 8.7 16 7.0 8.1 30 0.4 48

91 47 18 37 78 125 324 115 -25 7.0 I0 25 3.5 13 5.45.9 27 0 49

98 45 18 34 73 ! !4 336 108 -25 5.2 12 27 3.0 10.3 5.6 8.1 26 0 48

Human b GM37 skin

GM637

121 48 15 41 71 102 355 92 2.4 16 6.8 13 21 1.6 7.8 6.1 5.3 30 -46

116 53 18 43 76 90 340 92 -18 7.1 !6 27 5.4 7.7 8.0 17.6 19.0 0 47

I02 45 18 34 72 108 327 116 " -25 4.7 12 28 3.5 13 5.5 17.5 18 0 48

113 52 19 46 77 89 336 87 -20 5.8 14 26 6.3 7.8 11.0 8.7 34.4 0 47

a A s r e p o r t e d [10] b A s r e p o r t e d [ 11 ]

Table 2 H y d r o x y l y s i n e g l y c o s i d e s in p u r i f i e d t y p e I coilage~as f r o m n o r m a l a n d t r a n s f o r m e d f i b r o b l a s t s Cells

Glc-Gal-I~yl

Ga!--Hyl

Total substiluted Hyl

Normal human fibroblast (GM37)

1.16

0.65

1.81

6.3

22.3

SV40-Transform, ed

2.3

1.57

3.87

11.97

24.2

fibroblast (GM637) V a l u e s are p r e s e n t e d as r e s i d u e s p e r 100O r e s i d u e s o f a m i n o acids 50

U.nsubstituted Hyl

Glycosylation o f H y i (%)

Volume 85, number 1

FEBS LETTERS

f o r m e d ceils w e r e c o n v e r t e d i n t o c o l l a g e n m o l e c u l e s by a crude proco~lagen peptidase preparation (fig.3), indicating that transformed procoltagens are sensitive to procollagen pepfidase. [t is e v i d e n t f r o m t h i s c o n ~ - n u n i c a t i o n t h a t S V 4 0 virus t r a n s f o r m a t i o n o f h u m a n s k i n f i b r o b t a s t s r e s u l t s in a l t e r e d p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n s i n t y p e Ir p r o c o l l a g e n a n d p r o b a b l y also i n t y p e [ H p r o c o l t a g e n . Transformation has been known to cause morphol o g i c a l as w e l l as b i o c h e m i c a l a l t e r a t i o n s in e u k a r y o t i c ceils [ 8 ] . [ f b i o c h e m i c a l a l t e r a t i o n s d u e t o v i r u s t r a n s f o r m a t i o n m i m i c s o m e o f t h e c o n n e c t i v e t i s s u e diso r d e r s , t h e s e cells will m a k e a u s e f u l m o d e l f o r investigations of such metabolic disorders. Also, these Mterations in procoiiagen post-translational modificat i o n f o l l o w i n g virus t r a n s f o r m a t i o n m a y p l a y a r o l e in neoplasia and tumorogenesis.

Ackttow[edgements This r e s e a r c h w a s s u p r o r t e d b y g r a n t s f r o m N I H (HD09727), American Heart Association (74-740), a n d a Basil O ' C o n n o r S t a r t e r R e s e a r c h G r a n t f r o m the Nation~l Foundation-March of Dimes (5-59). R . L . C . is a r e c i p i e n t o f a R e s e a r c h C a r e e r D e v e l o p m e n t A w a r d f r o m t h e U S P H S . We t h a n k Bill F r e d e r i c k

January i 9 7 8

f o r e x c e l l e n t t e c h n i c a l a s s i s t a n c e . We also t h a n k Drs Ian Freeman and Jean-Paul Vergnes, Ophthalmology Research Laboratory, for amino acid and glycoside a n a l y s e s .

R e f e refaces [1] Layman, D. L., McGoodwin,.E.B. and Martin, G. R. (1971) Prec. Natl. Acad. Sci. USA 6 8 , 4 5 4 - 4 5 8 . [2] Smith, G. D., Byers, P. H. and Martin, G. R. (1972) Proc. Natl. Acad. ScL USA 68, 3260--3262. [3| Goldberg, B., Epstein, E. H., jr. and Nherr, C. J. (1972) Proc. Natl. Acad. ScL USA 69, 3655--3659. [4] Go~dberg, B. and Sherr, C. 3. (1973) Proe. Natl. Acad. ScL USA 70, 361--365. [5] Sherr, C. J., Taubnlan, M. B. and Goldberg, B. (1973) J. Biol. Chem. 248, 7033--7038. [6] Church, R. L. and Tanzer, M. L. (!975) FHBS Lett. 53, 105--109. [7] Goldberg, B., Taubmau, M. B. and Radin, A. (1975) Cell 4, 4 5 - 5 0 . [8] Tooze, J. (1973) The Molecular Biology. o f Tumor Viruses, Cold Spring Harbor Laboratory. [9] Church, R. L., Yaeger, J. A. and Tanzer, M. L. (1974) $. ~ o l . Biol. 86, 785--799_ [101 Fleischm~jer, R. and Fishman, L. (1965) Nature 205, 264. [ 11] Epstein, E. H. (1974) J. Biol. Chem. 249, 3 2 2 5 - 3 2 3 2 . [ 12] Kao, W. ~., Berg, R. A. and Prockop, D. J. (1976} Fed. Proc. Fed. Am. Soc. Exp. Biol. 35, 1739.

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