227

Biochimica et Biophysica Acta, 533 (1978) 227--237 © Elsevier/North-Holland Biomedical Press

BBA 37855

SIMILARITY OF ANTIGELATIN FACTOR AND COLD INSOLUBLE GLOBULIN

WALTRAUD. DESSAU, FRANZ JILEK, BERNHARD C. ADELMANN and HELMUT HORMANN A bteilung fiir Bindegewebsforschung, Max-Planck-Institut fh'r Biochemie, D-8033 Martinsried (G.F.R.)

(Received May 24th, 1977) (Revised manuscript received October 14 th, 1977)

Summary

Antigelatin factor, a protein capable of complexing denatured collagen, was separated from human serum by adsorption onto immobilized collagen. Anti- serum raised against the material binding to denatured collagen permitted the development of a radioassay for the determination of antigelatin factor in which the complex of antigelatin factor and denatured 12SI-labeled collagen is precipitated with this antiserum.

Further purification of antigelatin factor was achieved by chromatography on DEAE-cellulose yielding an electrophoretically homogeneous protein. Its migration rate in dodecyl sulfate-polyacrylamide gel electrophoresis was iden- tical with that of cold insoluble globulin (molecular weight approx. 440 000) prepared from human plasma by a published procedure amended by DEAE- cellulose chromatography. Reduct ion of disulfide bonds yielded subunits of molecular weight approx. 220 000, indistinguishable from those of cold insoluble globulin. The amino acid composit ion of both proteins was very simi- lar. Immunological identity of both proteins was demonstrated by gel diffusion against monospecific anti-cold insoluble globulin antiserum. Closely related binding curves were obtained if denatured '2SI-labeled collagen was reacted with increasing amounts of either cold insoluble globulin or antigelatin factor and the complexes formed were precipitated with anti-cold insoluble globulin antiserum. In addition, antigelatin factor and cold insoluble globulin mediated the fixation of denatured ~2SI-labeled collagen to trypsinized macrophages in the same way. Therefore, it is concluded that antigelatin factor and cold inso- luble globulin are identical or very closely related proteins.

Introduction

Several authors have described a serum protein capable of associating with gelatin, one of the denatured forms of collagen. Maurer found gelatin to pre-

228

cipitate with a number of human sera [1] and with the sera of a variety of ani- mal species [2]. Similar observations were made when a haemagglutination assay was used [3,4]. The nature of the reactive material remained uninves- tigated until Wolff et al. [5] found the activity in the euglobulin fraction of serum and provided evidence for the activation of this "antigelatin factor" by a second serum factor. Antigelatin factor appeared in the exclusion volume of Sephadex G-200 columns and was clearly separated from IgG [5]. Similar material was later detected by radioimmunoassay [6]. Attention was attracted to this substance when it was shown that macrophages could discriminate between native and heat-denatured collagen and that this reaction was mediated by a serum protein passively adsorbed to macrophages and possessing similar properties as antigelatin factor [ 7].

We now wish to report experiments which establish a close relationship between human antigelatin factor and cold insoluble globulin. This plasma pro- tein was detected by Morrison et al. [8] and was at first purified by Mosesson and Umfleet [9]. The data presented in this communication show that anti- gelatin factor and cold insoluble globulin are extremely similar with respect to biochemical and immunological properties.

Materials and Methods

Preparation of antigelatin factor. To 500 ml citric acid/sodium citrate/ dextrose-treated plasma in a plastic beaker were added: 10 000 kaUikrein inhi- bitor units Trasylol (Bayer, Leverkusen, G.F.R.), 100 N.I.H. units thrombin (Thrombinum purum, Behringwerke, Marburg, G.F.R.) dissolved in 1.0 ml 0.35 M CaCl:, pH 7.2, and 735 mg solid CaC12.2H20. After incubation at 37°C for 4 h the clot was separated from serum with the aid of a silk cloth.

The serum (100--200 ml) was applied to an affinity chromatography column (3 × 27 cm). The column contained heat-denatured calf skin collagen type I linked to Sepharose 4B (Pharmacia, Stockholm, Sweden). This absorbent had been prepared by the method of Cuatrecasas [10] and contained 1.5 mg col- lagen per ml packed gel. The column was operated in the cold room at an elu- tion velocity of 32 ml/h. After application of serum the column was washed with 750 ml 0.05 M Tris • HC1 buffer (pH 7.6)/0.1 M NaC1/0.025 M e-amino- caproic acid. Antigelatin factor was desorbed with 1.0 M KBr in 0.05 M Tris • HC1 buffer (pH 5.3)/0.025 M e-aminocaproic acid. The protein-containing frac- tions were dialysed against 0.039 M Tris/phosphate buffer, pH 8.6, and concen- trated by ultrafiltration (membrane UM 20 E, Amicon, Witten, G.F.R.) to an absorbance of approximately z~A1cm280nm = 1 . 0 .

Approx. 30 ml of this material was applied to a DEAF-cellulose column (2 X 15 cm, "Servacel DEAE 23 SS", Serva, Heidelberg, G.F .R. )equi l ibra ted with 0.039 M Tris/phosphate buffer, pH 8.6. The column was washed succes- sively with 0.039 M Tris/phosphate buffer, (pH 8.6), 0.127 M Tris/phosphate (pH 6.2), 0.220 M Tris/phosphate (pH 5.3) and 0.5 M Tris/phosphate (pH 4.2). All antigelatin factor activity was eluted at pH 5.3. The protein was dialysed against 0.1 M Tris • HC1 buffer (pH 7.4)/0.1 M NaC1 and stored frozen.

~ 2 8 0 n m = The extinction coefficient of cold insoluble globulin was assumed as =L0mg/ml 12.8 [11].

Cold insoluble globulin. This was prepared from human plasma as described

229

by Mosesson and Umfleet [9] and further purified by chromatography on DEAE-cellulose by the same procedure as antigelatin factor. The final product migrated as a single band in polyacrylamide gel electrophoresis.

Collagen. Collagen type I was extracted from fetal calf skin and purified as described [12]. Purity was verified by chromatography on carboxymethyl- cellulose [ 13] and by polyacrylamide gel electrophoresis [14] of the denatured molecule as well as by chromatography on DEAECellulose [15,16] of the peptides obtained by cyanogen bromide digestion [17] of the a-chains. The preparations were free of detectable non-collageneous contaminants. The triple- helical (native) conformation was converted into the random coil (denatured) conformation by heating collagen solutions in 0.01 M acetic acid for 60 min to 56 ° C. Collagen was labeled with l:sI as previously described [6].

Antisera. Goats were injected subcutaneously at multiple sites six times in weekly intervals with 0.15 mg antigelatin factor (eluate from affinity chro- matography) emulsified with Freund's complete adjuvant containing Micro- coccus butyricum (Difco, Detroit, U.S.A.). The animals received an additional injection 1 week later of 1.1 mg antigelatin factor in adjuvant and were bled after one more week and at various intervals thereafter. The immunization scheme was repeated as required. Some of these antisera produced an unaccept- ably high background when employed in the collagen binding assay. This could be remedied by passing such sera over denatured collagen linked to Sepharose and thus removing antigelatin factor present in goat serum.

Antiserum to cold insoluble globulin was prepared in rabbits using the mate- rial after DEAE-cellulose chromatography. The animals were injected sub- cutaneously with 1 mg protein in a total of 4 ml buffer and Freund's complete adjuvant twice at an interval of 3 weeks. They were bled 10 days later. An addi- tional monospecific antiserum to cold insoluble globulin prepared in rabbits was kindly donated by Behringwerke.

Collagen binding assay. Plastic tubes were charged with serial dilutions of antigelatin factor or serum (0.1 ml), 100 ng denatured 12SI-labeled collagen (in 0.1 ml 0.1 M Tris • HC1 buffer (pH 7.4)/0.1 M NaC1) and 0.1 M Tris • HC1 buffer (pH 7.4)/0.1 M NaC1/1% bovine serum albumin (0.2 ml). This was incu- bated in the cold overnight, then 0.2--0.5 ml antiserum to antigelatin factor was added and the incubation continued again overnight. The precipitate was collected by centrifugation and washed three times with 0.01 M Tris. HC1 buffer (pH 7.4)/0.1 M NaC1/l% bovine serum albumin. Controls contained 0.1 ml buffer instead of antigelatin factor solution. Radioactivity was deter- mined in a well-type scintillation spectrometer (Gamma 300, Beckman Instru- ments, Palo Alto, U.S.A.). Results were expressed as

Collagen bound (ng)= cpm(ppt,exptl) -- cpm(ppt,control) X 100 cpm(added)

Binding of antigelatin factor to trypsinized macrophages [7]. Guinea-pigs were injected intraperitoneally with 20 ml mineral oil (Paraffinum liquidum, DAB 7, Merck, Darmstadt, G.F.R.). Peritoneal exudate cells were harvested 3--5 days later. Washed cells were suspended in Hank's balanced salt solution ( 2 . 1 0 7 cells/ml) and treated with trypsin (Worthington TL7IR, 100 pg/107

230

cells) for 60 min at room temperature. The cells were washed once and resus- pended to 107 cells/ml. To 0.5 ml of this suspension was added 0.02--0.2 ml antigelatin factor solution. After incubation at room temperature for 60 min the cells were washed once with balanced salt solution and exposed to 100 ng denatured '2SI-labeled collagen in 0.1 ml balanced salt solution. The cells were again incubated at room temperature for 60 min and then washed thrice with balanced salt solution. Cell-bound '2SI-labeled collagen was determined in the gamma spectrometer.

Polyacrylamide gel electrophoresis. The method of Furthmayr and Timpl [14] was followed using 5% acrylamide and 0.2% sodium dodecyl sulfate. Sam- ples were dissolved in 0.01 M phosphate buffer (pH 7.0)/0.2% sodium dodecyl sulfate/5 M urea. They were reduced, if required, with dithioerythritol in excess for 60 min at 55°C. Between 6 and 12 pg protein were applied to indi- vidual gel cylinders (6 × 100 mm) and subjected to electrophoresis for 10 h at 6 mA per gel. Protein bands were stained with Coomassie Brillant Blue.

Gel diffusion. The method of Ouchterlony [18] was used in a microversion on microscope slides.

Amino acid composition. Samples (0.2 mg protein) were hydrolyzed in 6 M HC1 containing 0.05% mercaptoethanol for 12 h at 110°C under nitrogen and analyzed in a model D-500 amino acid analyzer (Durrum, Palo Alto, U.S.A.).

Results

Assay of antigelatin factor Antigelatin factor had originally been defined as a serum protein capable of

reacting with denatured collagen. We, therefore, developed an assay in which the binding of antigelatin factor to randomly coiled (denatured) ~2SI-labeled col- lagen is measured. The complex of the two proteins is soluble at physiologic ionic strength and pH, bu t it can be precipitated by antiserum from goats directed against a plasma fraction obtained by affinity chromatography on immobilized denatured collagen (antiantigelatin fator). Titration of antigelatin factor at constant '~SI-labeled collagen concentrations results in dose vs. effect relationships which resemble the sigmoidal curves frequently observed in true radioimmunoassays (Fig. 1, curve designated antigelatin factor). In the assay of antigelatin factor, however, the shape of the curves does not only reflect less binding of '2SI-labeled collagen by less antigelatin factor but the shift of the antigen/antibody ratio in the secondary reaction towards ant ibody excess as well. The assay is suitable in spite of this limitation for detecting antigelatin factor during purification steps and for comparing different antigelatin factor preparations at equal concentrations.

Antigelatin factor may not only be detected by its capacity to bind denatured collagen but in addition by its own affinity for trypsinized macro- phages. Macrophage-bound antigelatin factor in turn will associate with denatured collagen thus mediating the uptake of denatured collagen by these cells. This property of antigelatin factor was exploited in an assay in which trypsinized guinea-pig exudate cells were allowed to react with antigelatin fac- tor, washed, and exposed to denatured 125I-labeled collagen. Thus, ~2SI-labeled collagen in this assay served only as an indicator for the uptake of antigelatin

2 3 1

60

S 2o

40 CIG z~"

/ ~,/7 ~'AGF 20

o ~

" 2 3 4 5 6 kGF or CIG [1010gng]

Fig. 1. Binding of denatured 1 251.labele d collagen (1OO ng) to increasing amounts of antigelatin factor (© . . . . . . o) or cold insoluble globul in (m e) fo l lowed by p rec ip i t a t ion of t h e co mp lex es wi th anti- s e rum to ant ige la t in fac tor . Ant i -an t ige la t in fac tor was d i rec ted against a par t ia l ly pur i f ied an t ige la t in fac- tor p r epa ra t i on o b t a i n e d by adso rp t i on of s e r u m to i m m o b i l i z e d d e n a t u r e d col lagen. The same ant ige la t in f ac to r p repa ra t i on was used in the assay. Cold insoluble globul in was of final s tage pur i ty .

factor by the cells. Again, this procedure was useful for detecting antigelatin factor activity during isolation steps.

Preparation of antigelatin factor Antigelatin factor was usually prepared from serum. If plasma was permitted

to clot in the cold, the resulting serum bound less denatured 12SI-labeled col- lagen than serum prepared by clotting at 37°C (Table I).

Antigelatin factor could be separated from the bulk of serum proteins by affinity chromatography using heat-denatured calf skin collagen type I covalently linked to Sepharose. Binding of antigelatin factor to collagen occurred at nearly physiological ionic strength and pH, desorption was achieved with 1 M KBr at pH 5.3. It is apparent from Fig. 2 that all activity was bound to the adsorbent and that the material eluted with KBr was active in the col- lagen binding test as well as capable of "arming" macrophages for the uptake

T A B L E I

D E P E N D E N C E OF H U M A N S ERUM TO BIND D E N A T U R E D 1 2 5 I - L A B E L E D C O L L A G E N ON T H E T E M P E R A T U R E OF T H E S ERUM P R E P A R A T I O N

Plasma was p e r m i t t e d to c lot at e i ther t e m p e r a t u r e and 0.1 ml of the resul t ing se rum was r eac t ed wi th d e n a t u r e d 125i.labele d col lagen. The c o m p l e x f o r m e d was p rec ip i t a t ed wi th an t i s e rum to ant igela t in fac- tor. Bovine se rum a l bumin was subs t i tu ted for se rum in the con t ro l expe r imen t s .

T e m p e r a t u r e of se rum p r e p a r a t i o n

l 25 I- labeled collagen p rec ip i t a t ed (ng)

0.1 m l se rum Cont ro l *

4°C 27.0 9.0 33 .0 10.0

37°C 64.7 7.5 61 .5 7.7 4 7 . 8 5.3

* 0.1 ml b u f f e r con ta in ing 3% bov ine s e rum a lbumin . No prec ip i ta te is ob ta ined . The da t a r ep resen t r ad ioac t iv i ty adhe r ing to the tubes .

232

2.0-

1.5-

1.0-

0.5-

20 40 I0 20 30 FRACIION NUMBER

E

-3 000

-2000 N i

._a

1000

Fig. 2. Aff in i ty e h r o m a t o g r a p h y of h u m a n s e r u m on i m m o b i l i z e d d e n a t u r e d calf col lagen. Af te r applica- t ion of the sample the c o l u m n was washed wi th 0 .05 M Tris • HC1 (pH 7 .6) /0 .1 M NaC1/0.025 M e-amino- caproic acid. Ant ige la t in f ac to r was de so rbed wi th 0 .05 M T r i s . HCI (pH 5 .3) /1 .0 M K B r / 0 . 0 2 5 M e -aminocapro ic acid. The e f f luen t was m o n i t o r e d a t 280 n m (e - -o ) , and individual f rac t ions were tes ted in the collagen b ind ing assay (a A) and for the i r capac i ty to med i a t e the up t ake of d e n a t u r e d 125i_labele d collagen by t ryps in ized m a c r o p h a g e s (D •).

of denatured 12SI-labeled collagen. Further purification was accomplished by chromatography on DEAE-cellulose. Proteins were eluted from the ion- exchanger by stepwise increasing the ionic strength and decreasing the pH of the eluant. Pooled fractions were evaluated by polyacrylamide gel electrophore- sis (Fig. 3) as well as for their capacity to bind denatured 12q-labeled collagen and to mediate uptake of denatured 12SI-labeled collagen by macrophages (Table II). All active material was eluted with 0.220 M Tris/phosphate, pH 5.3. It migrated in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate as a prominent slow band. The yield in a typical experiment was 5.7 mg antigelatin factor from 50 ml serum. If antigelatin factor is assumed to be identical with cold insoluble globulin whose concentration in serum has been reported as 20 mg/100 ml [9], the recovery is 57% after this two-step purification procedure.

Comparison of antigelatin factor and cold insoluble globulin Antigelatin factor from serum and cold insoluble globulin prepared from

plasma had the same electrophoretic mobilities in sodium dodecyl sulfate con- taining polyacrylamide gels (Fig. 3). The molecular weight of cold insoluble globulin has been reported as 440 000 [11]. Reduct ion of antigelatin factor resulted in a single species of polypept ide chains displaying the same electro- phoretic properties as the two identical subunits of cold insoluble globulin whose molecular weight is 220 000 [11,19]. The minor bands visible in the electropherogram of unreduced antigelatin factor disappeared upon reduction of the sample.

The amino acid composit ions of antigelatin factor and cold insoluble globu- lin are very similar (Table III). They suggest, particularly when viewed in con- nection with the results of the electrophoresis, that the two proteins are either identical or very closely related.

233

S I II III C IIIr Cr Fig. 3. Po lyac ry l amide gel e lec t rophores i s in the p resence of sod ium d ed ecy l sulfate of f rac t ions ob ta ined by c h r o m a t o g r a p h y on DEAE-cel lu lose . Crude ant ige la t in fac tor p r epa red f rom se rum by aff ini ty chro- m a t o g r a p h y on i m m ob i l i z e d d e n a t u r e d col lagen (S) was appl ied to the DEAE-cel lu lose c o l u m n . Frac t ions (I, II , I I I ) were e lu ted by a s tepwise grad ien t as descr ibed in Materials and Methods . All an t ige la t in f ac to r act iv i ty was e lu ted in f rac t ion III. Af t e r r educ t i on , this f rac t ion mig ra t ed as a single band ( I n r ) . CIG (cold insoluble globul in) b o t h u n r e d u c e d (C) and r educed (Cr) is inc luded for compar i son .

Fig. 4. Gel d i f fus ion of cold insoluble globul in (CIG) and ant igelat in fac tor ( A G F ) against a monospec i f i c a n t i s e r u m to cold insoluble globul in showing c o m p l e t e ant igenic iden t i ty of cold insoluble globul in and ant ige la t in factor .

When antigelatin factor and cold insoluble globulin were set up in a gel dif- fusion experiment against monospecific antiserum to cold insoluble globulin, two single bands were observed which fused completely {Fig. 4). This identity of antigenic determinants was likewise apparent when the antiserum was directed to antigelatin factor (not shown).

Cold insoluble globulin, like antigelatin factor, was capable of binding denatured 12SI-labeled collagen, and the complexes formed could be precipi- tated with an antiserum to antigelatin factor (Fig. 1, curve designated CIG). Lack of congruence of the curves shown in Fig. 1 is probably due to different degrees of purity of the antigelatin factor and cold insoluble globulin prepara- tions employed. If, however, complexes were formed from denatured 12sI- labeled collagen and either cold insoluble globulin from plasma or antigelatin factor from serum, both proteins of final stage purity, and monospecific anti. cold insoluble globulin antiserum was used for immune precipitation, very similar binding curves were obtained (Fig. 5). Therefore, antigelatin factor

2 3 4

T A B L E II

C H A R A C T E R I Z A T I O N OF F R A C T I O N S O B T A I N E D BY C H R O M A T O G R A P H Y OF C R U D E A N T I - G E L A T I N F A C T O R ON D E A E - C E L L U L O S E

Ant ige la t in f a c t o r p r e p a r a t i o n f r o m a f f in i ty c h r o m a t o g r a p h y was appl ied to the c o l u m n and f r ac t ions ( I , I I J I I , I V ) were e lu ted by a s t epwise g r ad i en t o f inc reas ing ionic s t r eng th and dec reas ing pH as i nd i ca t ed . They were c o n c e n t r a t e d by u l t r a f i l t r a t i on to the abso rbanc i e s s t a ted and eva lua ted by the i r c apac i t y to b ind d e n a t u r e d 125 I-labeled col lagen and to m e d i a t e the u p t a k e of d e n a t u r e d 125 I- labeled co l lagen by m a c r o p h a g e s .

F r a c t i o n E lu t ion b u f f e r A 2 S 0 n m D e n a t u r e d 12Si . labe le d Cel l -bound

( T r i s / p h o s p h a t e ) col lagen b o u n d by 0.1 ml col lagen f r ac t i on (rig) ( c p m )

M pH

S ta r t ing ma te r i a l 0 .27 49 .5 1470

I 0 .039 8.6 0 .49 7.0 380

II 0 .127 6.2 0 .25 5.0 S00

I I I 0 .220 5.3 0.51 41 .0 1 2 5 0 IV 0 .500 4.2 0 .18 7.0 850 Con t ro l * 5.0 680

* 0.1 ml b u f f e r c o n t a i n i n g 3% bov ine s e rum a l b u m i n .

and cold insoluble globulin can bind denatured collagen comparatively well, and the resulting complexes can be precipitated effectively with a suitable anti- serum.

Finally, cold insoluble globulin and antigelatin factor were tested for their capacity to restore the binding capability of trypsinized macrophages for denatured '2SI-labeled collagen (Fig. 6). The binding curves of antigelatin factor and cold insoluble globulin very closely resembled each other and the amounts required for saturation were approximately the same. Obviously, cold inso- luble globulin can functionally replace antigelatin factor in this assay as well.

T A B L E I I I

A M I N O A C I D C O M P O S I T I O N OF A N T I G E L A T I N F A C T O R A N D C O L D I N S O L U B L E G L O B U L I N

A m i n o acid An t ige l a t in f ac to r Cold insoluble g lobu l in

Cys /2 3.2 3.8

Asp 9.5 9 .5

Ser 9 .5 10.0 T h r 9 .2 8.8 Glu 13.2 15.3 Pro 5.2 5.0

Gly 9 .0 9 .2

Ala 5.0 5.1 Val 6.9 7.7 Met 1.1 1.0

Ile 4,0 4.1 Leu 5,3 5.3 T y r 4,6 3.7 Phe 3.0 2.1 His 2.0 2.1 L y s 4 .2 3.0 Arg 5.1 4.4

235

= 6{

40

o

20

~'kGF

E

1500 AGF

I ooc

/ I I I I ~' I I

2 3 ~, 5 5'0 100 150 kGF or CIG llOlogng] PROTEIN [~g]

Fig. 5. Binding of d e n a t u r e d 125i . labeled collagen (100 ng) to increasing a m o u n t s of ant ige la t in fac tor (o . . . . . . o) or cold insoluble globul in (e e) fo l lowed b y p rec ip i t a t ion of the c o m p l e x e s wi th anti- s e rum to cold insoluble globulin. Ant ige la t in f ac to r and cold insoluble globulin were of final s tage pu r i ty .

Fig. 6. Binding of d e n a t u r e d 125I- labeled collagen to m a c r o p h a g e s a f te r " a r m i n g " the cells wi th anti- gelat in fac tor or cold insoluble globulin.

Discussion

Cold insoluble globulin is a plasma protein which represents the soluble form of a surface protein of fibroblasts [20] and has recently attracted considerable attention since one of its functions in the cell membrane is to mediate the at tachment of these cells to collagen and presumably to fibrin as well. The pro- tein is synthesized by fibroblasts, constantly shed into the interstitial space and can finally be detected in the blood [21]. A collagen-dependent fibroblast a t tachment factor from serum described by Klebe [22] has been found to con- tain cold insoluble globulin and its early plasminolytic split products [ 23 ]. This material is probably identical with a factor released from fibroblasts by plasmin [24]. Cold insoluble globulin isolated from plasma has a molecular weight of approx. 440 000 [11] and consists of two probably identical subunits of 220 000 [11,19]. The subunits are linked in the native molecule by disulfide bridges which are apparently located in terminal regions of the polypeptide chains. This port ion of the molecule is cleaved off during the early stages of plasminolytic attack and subunits are liberated [23].

The present investigation establishes an intimate relationship between cold insoluble globulin and antigelatin factor. Antigelatin factor has been described as a serum protein reacting with the random coil conformation of collagen in a similar manner as immunoglobulin [6]. It is clear from the data presented that antigelatin factor isolated from serum by this two-step purification procedure involving affinity chromatography on immobilized denatured collagen and chromatography on DEAE-cellulose is not an immunoglobulin but closely resembles cold insoluble globulin isolated from plasma by the method of Mosesson and Umfleet [9]. Antigelatin factor so isolated migrates in sodium dodecyl sulphate-containing polyacrylamide gels with the same electrophoretic mobili ty as cold insoluble globulin. Reduct ion of antigelatin factor and cold insoluble globulin gives rise to the apparently same subunits. The amino acid

236

compositions of antigelatin factor and cold insoluble globulin are extremely similar and distinctly different from any of the immunoglobulin classes. How- ever, the binding of denatured collagen to antigelatin factor resembles an antigen-antibody reaction in that antigelatin factor may be detected in serum not only by radioassay [6] but also by passive haemagglutination using erythrocytes coated with denatured collagen (Adelmann, B.C. and Timpl, R., unpublished}. Such systems are, therefore, not suitable for detection of anti- bodies to denatured collagen in human sera unless it can clearly be demon- strated that the reacting material is truely antibody and not antigelatin factor.

Similarity of antigelatin factor and cold insoluble globulin is further sub- stantiated by the identification of four functional sites whose properties are identical in antigelatin factor and cold insoluble globulin.

(a) Fibrin-binding site(s). The yield of antigelatin factor was comparably low when this protein was isolated from serum prepared in the cold and high when plasma was allowed to clot at 37°C. This may be explained by assuming that antigelatin factor is bound to the fibrin clot and that the association is more firIr, at 4°C than at 37°C. Ruoslahti and Vaheri [20] and Mosher [19] have similarly reported that cold insoluble globulin is bound to a fibrin clot in sub- stantial amounts in the cold but not at 37°C. Temperature dependence of the affinity of cold insoluble globulin for fibrin was also observed by Stemberger and HSrmann [25].

(b) Collagen-binding site(s}. Antigelatin factor as well as cold insoluble globulin can bind collagen in its random coil conformation in a dose-dependent manner (cf. Figs. 1 and 5). The amount of collagen bound at saturation is approximately equal for antigelatin factor and cold insoluble globulin.

(c) Antigenic determinant(s). Gel diffusion using antiserum to either anti- gelatin factor or cold insoluble globulin revealed complete antigenic identity of the two proteins. In addition, the complexes of denatured collagen and either antigelatin factor or cold insoluble globulin can be precipitated with anti- sera to antigelatin factor or antisera to cold insoluble globulin.

(d) Binding to macrophages. Guinea-pig antigelatin factor is present on the surface of guinea-pig peritoneal exudate cells [7]. It can be removed from these cells by trypsin [7] and such cells can be reconstituted with guinea-pig or human antigelatin factor as well as with human cold insoluble globulin. There- fore, antigelatin factor and cold insoluble globulin must both possess sites which are recognized by a receptor on the membrane of phagocytic cells. The affinity of cold insoluble globulin for macrophages and probably the usual presence of cold insoluble globulin on untreated macrophages is a hitherto unrecognized property of this protein. Antigelatin factor on the surface of macrophages is instrumental in the selective uptake of denatured collagen by these cells.

The data presented are insufficient to decide if antigelatin factor and cold insoluble globulin are identical proteins, if they represent different derivatives of a common parent molecule, or if one is derived from the other by loss of a small number of residues. Such alterations may have been caused by the actions of activated serum factors but they must be slight so as to not grossly affect molecular weights and functional sites.

It may be anticipated that the level of antigelatin factor/cold insoluble

237

globulin in body fluids such as plasma and synovial fluid is related to the occur- rence and the severity of disease processes involving the connective tissues and that determination of this level may prove useful for monitoring such pro- cesses.

Acknowledgements

The authors wish to thank Mrs. V. Jelini~, Mrs. E. Loibl and Mrs. M. Hable for their expert technical assistance and the Deutsche Forschungsgemeinschaft (SFB 51 and Ad 41/1 ff.) for supporting these investigations.

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