An assay for Clq biosynthesis in cultured human fibroblasts

Journal of Immunological Methods, 56 (1983) 43-54 Elsevier Biomedical Press

43

An Assay for C lq Biosynthesis in Cultured Human Fibroblasts K.A. F l e m i n g , K . B . M . R e i d ~ a n d J . O ' D . M c G e e * University of Oxford, Nuffield Department of Pathology, John Radcliffe Hospital, Oxford, and I Department o/Biochemistry, South Parks Road, Oxford, U.K. (Received 4 March 1982, accepted 30 June 1982)

With a standard CI haemolytic assay, cultured human fibroblasts were shown to synthesize and secrete haemolytically active CI and Clq. An assay for detection and quantitation of intra-cellular biosynthesis and secretion of C l q was then developed, using Sepharose beads covalently coated with goat monospecific anti-C I q IgG. The molecular structure of fibroblast C Iq was found to be unusual in 2 ways: firstly, it probably represented an enlarged pro-CIq, and secondly, the secreted molecule had a different structure from the cell associated molecule. Key words: cultured human fibroblasts - - Cl q - - biosynthesis - - immunoassay - - molecular structure

Introduction

The first component of complement (CI) is a calcium dependent macromolecular complex of 3 protein sub-components, Clq, Clr and Cls (Mttller-Eberhard, 1975). The glycoprotein Clq initiates activation of the CI complex, by recognizing and binding to the Fc region of aggregated immunoglobulin of certain classes (Fothergill and Anderson, 1978). The molecular structure of Clq is partially similar to that of collagen (Reid and Porter, 1976), and it has been suggested that the Clq structural gene may be part of the collagen gene family (Solomon, 1980). Since fibroblasts synthesize Clq and collagen in vivo (AI-Adnani and McGee, 1976) and in vitro (Reid and Solomon, 1977), the possibility that their biosynthesis is regulated by similar mechanisms may be investigated in cultured fibroblasts. Prerequisites for such an investigation are sensitive assays for the biosynthesis of both proteins. Collagen synthesis can be measured by the collagenase assay (Peterkofsky and Diegelmann, 1971) but an assay for measurement of newly synthesized intra-cellular Clq has not been described; previous in vitro studies of Clq biosynthesis measured only secreted functional Ciq which accumulated in the tissue culture medium (Reid and Solomon, 1977; Morris et * To whom reprint requests should be addressed. 0022-1759/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press

44 al., 1978; Mi~ller et al., 1978a, b; Morris and Paz, 1980). This paper describes development of an assay for measuring Clq biosynthesis. Since there is evidence that the Clq synthesized in vitro differs from that found in serum (Reid and Solomon, 1977; Morris et al., 1978), a study of the nature of the Clq synthesized by cultured fibroblasts is also reported.

Materials and Methods

Human embryonic skeletal parts were obtained from vacuum terminations of pregnancy, and out-dated plasma from the Regional Blood Transfusion Service. Normal human serum was collected from healthy volunteers. Guinea pig serum was obtained from Olac Ltd. (Bicester, Oxfordshire), sheep red blood cells in modified Alsever's solution from Tissue Culture Services (Slough, England), or Flow Laboratories (Irvine, Scotland), all tissue culture reagents and plastic culture flasks from Gibco-Biocult (Paisley, Scotland), and radiolabelled amino acids from the Radiochemical Centre (Amersham, England); the latter included an amino acid mixture containing equal quantities, by activity, of: L-[4,5-3H]leucine (spec. act. 150 Ci/mmot), L-[4,5-3H]lysine (spec. act. 77 Ci/mmol), L-[2,4,6-3H]phenylalanine (spec. act. 65 Ci/mmol), L-[2,3,4,5-3H]proline (spec. act. 117 Ci/mmol) and L-[2,3,5,63H]tyrosine (87 Ci/mmoi). The concentration of these radiolabelled amino acids was 1 mCi/ml. Collagenase type III, from Clostridium histolyticum, was obtained from Sigma Chemical Company, Poole, and further purified in this laboratory to remove non-specific protease activity (Peterkofsky and Diegelmann, 1971); the purified enzyme had specificity only for molecules with collagen sequences. All other chemicals were of the highest purity available and were obtained from either B.D.H. or Sigma Chemical Co., Poole, or Hopkins and Williams, Romford. Cyanogen bromide activated Sepharose 4B (Pharmacia Fine Chemicals, Uppsala) was coupled according to the manufacturer's instructions to ovalbumin, so that approximately 10 mg ovalbumin was bound to 1 ml Sepharose beads. Similarly goat anti-CIq lgG (see below) was bound to Sepharose beads so that 1 ml beads contained approximately 5 mg anti-Clq. The Sepharose beads were then suspended at 10% (v/v) in assay buffer (see below) and stored at 4°C till used.

Preparation of Clq Clq was prepared from out-dated human plasma (Reid, 1974). The Clq was shown to be pure on 5.6% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) with or without reduction with 20 mM dithiothreitol, and on testing by double immunodiffusion against a rabbit anti-Clq monospecific antiserum.

Preparation of antibodies and immune aggregates Antibody to Clq was prepared in goat and rabbit by intra-muscular or sub-cutaneous injection of purified Clq with equal volumes of Freund's complete adjuvant. Whole IgG anti-Clq was prepared by standard methods (Hudson and Hay, 1976).

45

Washed insoluble immune aggregates of ovalbumin and either whole IgG or F(ab') 2 anti-ovalbumin were prepared at equivalence in phosphate-buffered saline (pH 7.2). The protein concentration of the precipitation was determined by the method of Lowry et al. (1951) with bovine serum albumin as a standard. All the antisera and antibody preparations were monospecific on double immunodiffusion and immunoelectrophoresis and were more than 90% pure on 5.6% SDS-PAGE, with or without reduction with dithiothreitol.

Cell culture Fibroblast cell lines were prepared by trypsin disaggregation of the skeletal parts of vacuum termination embryos. The cells had a stellate morphology, produced reticulin fibres and synthesized more than 10% of their total protein as collagenase degradable protein. They were grown as monolayers at 37°C, in air, in plastic flasks, in Eagle's minimum essential medium, containing Earle's salts, 20 mM Hepes (pH 7.4), L-glutamine (2 raM), penicillin (100 U/ml) streptomycin (100/~g/ml), fungizone (250 /~g/ml), sodium ascorbate (50 ttg/ml), and ferric nitrate (10 /~g/ml). Immediately before use, foetal calf serum was added to 10% (v/v) by filtration through a 0.45 /zm Millipore filter. The cells were grown in 1 ml medium/5 cm2 surface area of culture vessel, and the medium renewed every 4 days. The cultures were divided every 7 days by trypsinisation and plated at 2 × 104 ceils/cm 2. At this density the doubling time was 24 h, and confluence was reached by 5 days, when the number of cells was approximately 12 × 104/cm 2. Approximately every third passage, the cells were grown in antibiotic free medium.

Detection of C1 and Clq haemolytic activity Twenty-four hours after seeding, the medium was discarded and the cell layer washed 3 times with serum-free medium, l m i / c m 2 surface area. Fresh medium, containing foetal calf serum, 10% (v/v) was added. The foetal calf serum had been heat inactivated at 56°C, for 30 rain, then stored at - 2 0 ° C till use. An excess of 1 ml medium was added, and after incubation for 20 rain at 37°C, l rnl medium was withdrawn and kept at 37°C for the duration of the experiment. This sample was used for estimation of the initial amount of haemolytic activity present. At the end of the experiment the medium was collected and stored at 4°C till assayed (usually within 24 h). C1 haemolytic activity was assayed with sheep erythrocytes coated with antibody and the 4th component of complement (EAC4) according to Reid et al. (1977), and expressed either as a percentage of complete lysis or as the concentration of effective Ci molecules (era) (Rapp and Borsos, 1970).

Detection of Clq synthesis (1) Conditions for radiolabelling cells. For radiolabelling experiments, replicate 25 cm 2 flasks were used for each experimental variable. Flasks were seeded and cultured as described above. At the appropriate phase of growth the medium was removed and the cell layer washed 3 times with serum free medium, 1 ml/25 cm2 culture area. Then fresh medium with or without serum 10% (v/v) was added. Normally serum-free medium was used during the pulse period. The mixture of five

46 3H-labelled amino acids of high specific activity (4 # C i / m l of culture medium) and [3H]glycine ( 2 / ~ C i / m l of culture medium) were then added. The pulse period was usually 3 h. In experiments in which haemolytic activity was being measured also, heat-inactivated foetal calf serum was used. (2) Harvesting of radiolabelled cell layer and medium. All procedures were carried out on ice, except where otherwise stated. At the end of the pulse period, the medium was collected and the cell layer washed with ice cold phosphate-buffered saline (pH 7.2) containing 0.05% sodium azide ( w / v ) (I m l / 2 5 cm 2 culture area). The medium and the wash from replicate flasks were pooled. A 1/10 volume of 75 m M T r i s - H C l (pH 7.6) containing 50 mM sodium chloride, 200 mM EDTA, 1% ( v / v ) Triton X-100, 0.05% ( w / v ) gelatin, and 0.01% ( w / v ) sodium azide was added to the medium. The cell layer was washed once more with phosphate-buffered saline as above, then scraped off into 2 m l / 2 5 cm 2 culture area of the same buffer. The cell layer from duplicate 25 cm 2 flasks was pooled. After centrifugation at 500 x g for 5 min at 4°C, the cells were resuspended in assay buffer, i.e., 75 mM T r i s - H C ! (pH 7.6) containing 50 mM sodium chloride. 10 mM EDTA, 0.05% ( w / v ) gelatin. 0.1% ( v / v ) Triton X-100 and 0.01% ( w / v ) sodium azide (1 m l / 2 5 cm 2 culture area). The cell layer and medium were sonicated for 10 sec, at 7/.tm. Ribonuclease, 1 m g / m l in assay buffer, was added to both cell layer and medium at 20 t t g / I x 10 6 cells. After incubation at 37°C for 10 rain, 500 ~1 cell layer was taken for D N A estimation and stored at - 2 0 ° C till assayed. The remainder of the cell layer and medium were centrifuged at 1000 x g for 10 min at 4°C, and the supernatants stored at 4°C till assayed. This was usually done immediately, but always within 24 h. (3) Assay for Clq synthesis (solid-phase immunoassa.v). Duplicate tubes containing 35 ~1 radiolabelled cell layer or 300/~1 medium were made up to 1.0 ml with assay buffer. Sepharose ovalbumin and Sepharose anti-Clq (100/xl) were added, and the mixture incubated with rolling at 37°C for 18 h. After incubation, the beads were washed 3 times with 2 ml assay buffer (by rolling at room temperature for 10 min, followed by centrifugation at 1000 x g for 5 min at 4°C). They were then resuspended in 500 ~1 assay buffer which was then added to 10 ml NE 260 scintillant fluid. A further wash with 500/xl assay buffer was added to the scintillant fluid and the suspension counted. The amount of radiolabelled C l q was taken as the difference between radioactivity bound to the Sepharose ovalbumin beads and that bound to the Sepharose anti-Clq beads. C l q synthesis was then expressed as counts per minute ( c p m ) / m g D N A of each culture. Total protein synthesis was measured as radioactivity incorporated into trichloroacetic acid (TCA) insoluble material as described by Peterkofsky and Diegeimann (1971). The D N A content of the cultures was measured in an aliquot of the cell layer by the diphenylamine method of Burton (1956), with calf thymus D N A as standard. Results

C l q specificity of fibroblast medium haemolytic activity C I haemolytic activity was detected in fibroblast medium as described in Materials and Methods. This haemolytic activity was antibody dependent and was in-

47 TABLE I Clq SPECIFICITY OF FIBROBLAST MEDIUM HAEMOLYTIC ACTIVITY One hundred microlitres of fibroblast medium (5.5 x 10 s em/ml CI activity) were incubated in duplicate, with 100 tal EACA (i × 10S/ml) for 20 rain, at 30°C, to allow fibroblast CI activity to bind to the cells. After centrifugation, the cells were washed once in i ml ice cold buffer, with or without l0 mM EDTA. They were then resuspended in either 500 pl 'CIr, Cls reagent' or 500 ~tl buffer. CI activity was then assayed. As a control, 3.6 pg pure CIq was substituted for fibroblast medium. The 'Clr, CIs' reagent consisted of suitably diluted fibroblast medium. It was found that increasing dilution of fibroblast medium resulted in loss of detectable whole CI activity. Addition of pure Clq to this diluted medium restored whole CI activity, indicating not only that CIr, Cls activity remained, but also that it was present in greater quantities than CIq activity. Addition to EAC4 ceils

CI activity (% lysis)

Fibroblast medium Fibroblast medium, then EDTA wash Fibroblast medium, then EDTA wash then 'Clr, Cls' 'Clr, CIs' C I q + ' C I r , Cls'

66.9 12.6 19.1 9.0 87.3

h i b i t e d b y p r e - i n c u b a t i o n w i t h i n s o l u b l e w h o l e I g G i m m u n e aggregates, b u t n o t by i n s o l u b l e F(ab')2 i m m u n e aggregates. It was sensitive to h e a t i n a c t i v a t i o n at 5 6 ° C for 30 m i n a n d to i n c u b a t i o n w i t h 5 m M d i - i s o p r o p y l f l u o r o p h o s p h a t e . T h e h a e m o l y t i c a c t i v i t y a c c u m u l a t e d in t h e f i b r o b l a s t m e d i u m in a t i m e - d e p e n d e n t m a n n e r , a n d was i n h i b i t e d b y c y c l o h e x i m i d e . T h e s e c h a r a c t e r i s t i c s ( d a t a n o t s h o w n ) w e r e s i m i l a r to t h o s e d e s c r i b e d p r e v i o u s l y for f i b r o b l a s t m e d i u m C I h a e m o l y t i c a c t i v i t y ( R e i d a n d S o l o m o n , 1977). I n a d d i t i o n , t h e f i b r o b l a s t m e d i u m was a b l e to a c t i v a t e C l r , C l s . T h i s is s h o w n in

TABLE I1 COLLAGENASE DIGESTION OF FIBROBLAST MEDIUM CI HAEMOLYT1C ACTIVITY Double dilutions of 100 #1 purified bacterial collagenas¢, (50 #g/ml 50 mM Tris-HCI (pH 7.6) containing 5 mM calcium chloride) were made from 1 : 2 to 1 : 16, in 500 ~1 buffer. Fibroblast medium, 250/~1 (I.5× l0 s em/ml C1 activity) and N-ethyl malcimide to a final concentration of 2.5 raM, were added. The final volume made up to 1 ml with buffer. The mixture was incubated at 37°C for 1.5 h, then 500 #1 assayed in duplicate for CI activity. Addition to fibroblast medium

CI activity (c/, lysis)

Ni I Collagenas¢ (pg): 0 (buffer only) 5 2.5 1.25 0.625 0.32

86 76 17.1 27.6 24.6 40.5 46.7

48 TABLE Ill INHIBITION OF NORMAL HUMAN SERUM Clq HAEMOLYTIC ACTIVITY BY SEPHAROSE OVALBUMIN AND SEPHAROSE ANTI-Clq Sepharose ovalbumin and Sepharose anti-CIq beads (10 ml, 10% (v/v) suspension) were washed 3 times with 10 ml of phosphate-buffered saline (pH 7.2) containing 10 mM EDTA. Normal human serum, 8 ml. was dialyzed overnight at 4°C against 200 vols. of phosphate-buffered saline (pH 7.2) containing 10 mM EDTA, and 1 ml was added to the Sepharose beads. The suspension was incubated with rolling at 4°C h~r 20 h, and the supernatant collected. The beads were washed twice with 2 ml phosphate-buffered saline (pH 7.2) containing 10 mM EDTA, and these washes were added to the original supernatant. Double dilutions of supernatants were made in buffer and assayed for Clq dependent haemolytic activity according to the method of Kolb et al. (1979). Addition

Sepharose ovalbumin Sepharose anti-Clq Sepharose anti-CIq then Clq

Normal human serum haemolytic activity (% lysis) 6.25 ttl

12.5 t~l

25 ~tl

64.3 1.0 -

92.5 7.7

100 10.6 100

Table I. Addition of functionally pure ' C l r , C l s ' reagent to E A C 4 cells, which had been pre-incubated in fibroblast medium and then washed in 10 m M E D T A , increased the whole CI activity of the ' C l r , C l s ' reagent from 9% to 19.1% iysis, although this effect was not as marked as that obtained by pre-incubating the E A C 4 cells with purified C l q (87.3~ lysis). Purified collagenase digests collagenous structures such as C lq (Reid and Porter, 1976). Its effect on fibroblast medium CI haemolytic activity is seen in Table II. Although incubation of fibroblast medium with collagenase buffer slightly reduced the C I activity from 86% lysis to 76% lysis, collagenase destroyed the CI activity in a dose dependent manner, 2.5/~g collagenase producing 80% inhibition.

Assay for Clq synthesis Preliminary experiments established that the o p t i m u m conditions for detecting C l q synthesis were as described in Materials and Methods.

Specificity (a) Inhibition of Clq haemolytic activity by Sepharose beads. C l q haemolytic activity was removed only by Sepharose a n t i - C l q beads and not by Sepharose ovalbumin beads. This was shown for normal h u m a n serum by incubating both sets of Sepharose beads with serum (Table III), and then measuring residual haemolytic activity, After incubation with Sepharose ovalbumin, much complement dependent haemolytic activity remained, 25 /~1 supernatant giving 100% lysis. However, little remained after incubation with Sepharose anti-Clq, 25 /~1 supernatant giving only 10.6% lysis. Addition of purified C l q to the supernatant of the Sepharose a n t i - C l q beads restored 100% lysis, thus indicating that only C l q had been removed (Table

49

Co~lacjen BSA Ovalbumln

MyocjIoblr

15

(::)

10

~< ¢.J

50

100

Migration Distance (m rn)

Fig. 1. SDS polyacrylamide gel electrophoresis of radiolabelled material bound to Sepharose ovalbumin and Sepharose anti-Clq beads after incubation with cell layer. Reduced and alkylated material bound to Sepharose anti-Clq was digested with and without 5/~g purified collagenase. The maIerial bound to the Sepharose ovalbumin beads was reduced and alkylated only. Equal volumes of cell layer were incubated for each variable. The markers were run under reduced or non-reduced conditions where appropriate. Sepharose ovalbumin (5832 TCA cpm appfied) . . . . . . ; Sepharose anti-Clq, unreduced (9520 TCA cpm appfied) ; Sepharose anti-Clq (19,665 TCA cpm applied) • •; Sepharose anti-CIq, collagenase digested (18,450 TCA cpm applied) O . . . . . . O.

III). A similar effect was found using fibroblast medium as a source of C1 haemolytic activity. The concentration of effective molecules of C1 haemolytic activity left in fibroblast medium after incubation with Sepharose ovalbumin (3.6 × 108 e m / m l ) , was the same as that present in fibroblast medium incubated without Sepharose beads. However, the a m o u n t present in the medium incubated with Sepharose a n t i - C l q was 2 × 108 e m / m l ) , a fall of approximately 44%.

(b) SDS polyacrylamide gel electrophoresis of radiolabelled fibroblast material bound to Sepharose beads. Sepharose ovalbumin and Sepharose a n t i - C l q beads were incubated with radiolabelled cell layer and medium, after which 7.5% SDS polyacrylamide gel electrophoresis of the b o u n d material was carried out to determine its specificity and nature (Figs. 1 and 2). N o major peak of radioactivity b o u n d to the Sepharose ovalbumin beads from either cell layer or medium (Figs. ! and 2). Without reduction, the material b o u n d to the Sepharose a n t i - C l q beads from the

50 Collagen BSA

Ovalbumin

My~lo~in

al a2

1

6

,

5 o x

~4 o

2

1

0

°"o.O. I

A

Migrat,

I

J

I 50

on

D, stance(mm)

100

Fig. 2. SDS polyacrylamide gel electrophoresis of radiolabelled material bound to Sepharose ovalbumin and Sepharos¢ anti-Clq beads after incubation with medium. Reduced and alkylated material bound to Sepharose anti-Clq was digested with and without 5 #g purified collagenase. The material bound to Sepharose ovalbumin was reduced and alkylated only. Equal volumes of medium were incubated for each variable. The markers were run under reduced or non-reduced conditions where appropriate. Sepharose ovalbumin (1416 TCA cpm applied) . . . . . . ; Sepharose anti-Clq, unreduced (3264 TCA cpm applied) - - , Sepharose anti-CIq (5274 TCA cpm applied) • 0; Sepharose anti-Clq, collagenase digested (5190 TCA cpm applied) © . . . . . . ©.

cell layer also produced no major peak of radioactivity. However, on reduction, 2 major peaks were present (Fig. 1). These ran in a molecular weight range of 55,000-47,000 and 43,000-38,000. Although the 55,000-47,000 peak was usually present in greater quantities than the 43,000-38,000 peak, on occasion the larger molecular weight peak was missing, and a peak at 25,000 appeared. Presumably this reflected proteolytic degradation. On collagenase digestion, both peaks were reduced in a m o u n t and the molecular weight ranges d r o p p e d to 51,000-44,000 and 41,000-37,000. Since this was such a small decrease, this manoeuvre was repeated 4 times but with the same results. This indicates that the b o u n d material had some collagen•us structure. Although the material b o u n d to the Sepharose a n t i - C l q beads from the medium only showed a small peak of radioactivity (range 68,000-58,000) without reduction, on reduction a much larger single major peak was seen. This ran in the range 60,000-52,000 (Fig. 2). On collagenase digestion this peak was reduced

51 in amount and dropped in molecular weight to 56,000-47,000. Three smaller peaks of radioactivity appeared between 38,000-29,000. As in the cell layer, this small molecular weight difference following collagenase digestion was consistent over 4 experiments, and also indicates that the bound material had some collagenous structure.

Discussion

Investigation of the relationship between collagen and Clq biosynthesis by fibroblasts in vitro requires an assay capable of measuring synthesis of Clq in both cell layer and medium. As the first step in development of this assay, it was necessary to demonstrate that cultured fibroblasts indeed synthesize Clq. Since complement components are defined by their ability to act in concert with other complement components to produce haemolysis (Fothergill and Anderson, 1978), synthesis of haemolytically active Clq was shown in 3 ways. Firstly, fibroblast medium was found to contain CI haemolytic activity, which depended not only on the presence of antibody, but was absorbed by the Fc region of the IgG molecule, a highly specific property of Clq (Fothergill and Anderson, 1978). These findings are similar to those described by Reid and Solomon (1977). Secondly, the presence in fibroblast medium of the Clq function, activation of Clr, Cls (MOller-Eberhard, 1975), was shown using EDTA. Since 10 mM EDTA splits the Ca 2+ dependent CI macromolecular complex into its component parts, Clq, Clr and Cls, washing EAC4 cells, to which CI is bound, with EDTA, removes the Clr and Cls, but allows the Clq which is bound to antibody to remain. This Clq is then capable of further activating Clr, Cls. Fibroblast CI haemolytic activity bound to EAC4 ceils was indeed removed by washing in 10 mM EDTA, and 'Clr C ls reagent' incubated with these EDTA washed cells, was partially activated, as evidenced by the generation of whole C 1 activity. Thus, fibroblast medium displayed the Clq function, activation of Clr, Cls. The third method of confirming that medium from fibroblast cultures contained Clq haemolytic activity was by demonstrating that fibroblast medium Cl activity was susceptible to collagenase. Since Clq is one of the very few proteins with a collagenous structure, it is degraded by collagenase and its haemolytic activity is lost (Reid and Porter, 1976). Incubation of fibroblast medium with bacterial collagenase, purified to have specificity against collagenous proteins only, resulted in loss of Cl haemolytic activity, thus again indicating the presence of Clq activity in the fibroblast medium. De novo synthesis of the Cl haemolytic activity by the cultured fibroblasts was suggested by its accumulation in the medium, and this was confirmed by reversible inhibition of this accumulation by cycloheximide (results not shown). The rate at which C1 haemolytic activity accumulated in the medium was about 15 em per cell per hour, similar to that reported previously for fibroblasts (Reid and Solomon, 1977; Morris et al., 1978). Having confirmed that the cultured fibroblasts synthesized haemolytically active Clq, we developed an assay for measuring the newly synthesized Clq in both cell

52 layer and medium. This was a solid-phase immunoassay, consisting of Sepharose beads coated with monospecific goat anti-Clq IgG, and Sepharose beads coated with ovalbumin as a control. The specificity of the assay for Clq was shown in 2 ways. First, since complement components are defined by their haemolytic properties, the ability of the Sepharose beads to bind Clq haemolytic activity was tested. Incubation of fibroblast medium containing CI haemolytic activity with the Sepharose beads, showed that only the Sepharose anti-Clq beads removed C I haemolytic activity. The specificity of the Sepharose anti-Clq beads for C lq haemolytic activity was shown by removal of Clq dependent haemolytic activity from normal human serum. The second method of demonstrating the specificity of the assay for CIq was by SDS polyacrylamide gel electrophoresis of the radioactive material bound to the beads after incubation in pulsed labelled fibroblast cell layer and medium. This showed that the Sepharose ovalbumin beads bound little radioactive material from either cell layer or medium. However, the Sepharose anti-Ciq beads bound radiolabelled material from both cell layer and medium which was collagenase sensitive, and thus had some collagenous structure. Only 4 types of protein have been described as having a coilagenous structure: the interstitial and basement membrane collagens (Solomon, 1980), part of the acetylcholinesterase of the electric organ of the eel, Electrophorus electricus (Rosenberry and Richardson, 1977), a glycoprotein isolated from lung by alveolar lavage (Bhattacharyya and Lynn, 1980) and CIq (Reid and Porter, 1976). The material bound to Sepharose anti-Clq beads had an entirely different molecular weight to all known collagen chains, except some of the lower molecular weight collagen components extracted from basement membranes (Chung et ai., 1976). However, the molecular weight of the smallest of these collagens appears to be at least 70,000, since those smaller components originally described with a molecular weight in the order of 50,000 are now thought to be degradation products of larger molecules (Kresina and Miller, 1979). The lung alveolar glycoprotein (M r 36,000) and the collagenous subunits of the acetylcholinesterase (M r 44,000 and 40,000) have molecular weights which are not the same as those described here. Accordingly the specific binding of collagenase sensitive radiolabelled material (from cultures known to be synthesizing haemolytically active C l q) by Sepharose anti-Clq beads, which specifically bind C lq haemolytic activity, strongly suggests that the radiolabelled material bound to the Sepharose anti-Clq beads was a 'Clq-like' molecule. This being so, the assay may be used for measuring synthesis of Clq. The specificity of this assay for CIq is rather unusual since it has 2 independent components. Firstly, as in other immunoassays, the antigen recognition and binding site at the amino-terminal end of the anti-Clq IgG molecule presumably specifically recognizes and binds to an antigenic site or sites on the Clq molecule. Secondly, since coupling of the anti-CIq IgG molecules to the Sepharose beads effectively aggregates these molecules, Ciq, by virtue of its Fc recognition and binding function, specifically recognizes and binds to the Fc region of the aggregated IgG molecules. This gives this Clq assay a second and unique mode of specificity. It was for this latter reason that non-immune IgG Sepharose was not used as a control. The molecular structure of fibroblast Clq is unusual for 3 reasons. First, on SDS

53 polyacrylamide gel electrophoresis, while the material isolated from the medium migrates as 1 peak, that from the cell layer migrates as 2 peaks. Also, the molecular weight of the material from the medium is slightly larger (60,000-52,000) than that of the cell layer, which migrates between M r 55,000-47,000 and M r 43,000-38,000. This is contrary to the expected state of affairs, where the intracellular form of a secreted protein is usually larger, if anything, than the extra-cellular form. One explanation may be that the intra-cellular material represented an intermediate form of the secreted protein, and that post-translational modifications occurred before secretion. Another explanation may be that the extra-cellular and intra-cellular forms of Clq represented separate proteins, the intra-cellular protein corresponding to a membrane, perhaps receptor form of Clq, and the extra-cellular form corresponding to secreted Clq. This has been reported for IgM/~ chains synthesized by human lymphoma derived cell lines (Singer et al., 1980). However, in contrast to the situation found here, the membrane form of IgM # chain has a larger polypeptide chain than the secreted form. The second unusual finding was that while, on SDS polyacrylamide gel electrophoresis, unreduced native Clq migrates as 2 peaks of M r 69,000 and 54,000, the A - B and C - C chain dimers respectively (Reid and Porter, 1976), virtually no unreduced fibroblast Clq entered the gel. Since on reduction fibroblast Clq does enter the gels, it thus appears that the majority of the fibroblast Clq has additional disulphide linkages as compared with native human Clq. In addition, the molecular weights of its reduced components (between 60,000 and 40,000) are approximately 20,000 larger than native Clq, the A, B and C chains of which have a molecular weight of 31,600, 27,000 and 23,500 respectively on SDS-PAGE. Although on occasions, smaller molecular weight material was present in fibroblast Clq gels, these are interpreted as representing degradation products, since it was found that after storage, all the material migrated between M r 38,000 and 17,000, the majority being about 17,000 (results not shown). The third unusual finding was the size of the reduction in molecular weight induced by coUagenase digestion. In native Clq, the collagenase degradable region of each chain is approximately 10,000 molecular weight. That found in fibroblast Clq was approximately 4200-2000 in size. Prolonging the period of collagenase digestion to 24 h produced no further degradation. A possible, indeed probable, explanation for the above differences from native Clq is that the fibroblast Clq represented an enlarged 'pro' form of Clq. Synthesis of a 'pro-Clq' by cultured cells has been described previously, for fibroblasts (Reid and Solomon, 1977; Morris et al., 1978), and for bladder epithelium and monocytes (Morris et al., 1978), but the molecular weight and structure of these 'pro-Clqs' differed both from each other, and from that described here. However, the intracellular material described here (2 components of molecular weight between 55,000-38,000) resembles rather closely the medium 'pro-Clq' described by Reid and Solomon (1977). The presence of disulphide linked peptide extensions in a 'pro-Clq' could explain the unexpectedly small decrease in molecular weight found after collagenase digestion, since they may hinder digestion of the collagenous region. By analogy, it is known that. although native Clq takes up to 24 h to be

54 c o m p l e t e l y d i g e s t e d by c o l l a g e n a s e , o n r e m o v a l o f the n o n - c o l l a g e n o u s r e g i o n by p e p s i n d i g e s t i o n , the r e s i d u a l c o l l a g e n o u s r e g i o n c a n be d i g e s t e d by c o l l a g e n a s e w i t h i n 1 rain ( B r o d s k y - D o y l e et al., 1976). T h e r e a s o n s for the d i s c r e p a n c i e s b e t w e e n the m o l e c u l a r w e i g h t a n d s t r u c t u r e of the ' p r o - C l q ' d e s c r i b e d h e r e a n d that d e s c r i b e d in p r e v i o u s r e p o r t s are n o t clear, b u t o n e o b v i o u s d i f f e r e n c e is that the cells a n d c u l t u r e c o n d i t i o n s used are d i f f e r e n t .

Acknowledgements T h i s w o r k was Pharmaceuticals.

partially

supported

by

the

Weilcome

Trust

and

Janssen

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