25,000 to 56,000), shark cartilage chondroitin-6-sulfate (M, = 40,000 to 80,000), porcine skin dermatan sulfate, and heparan sulfate were obtained as previously ...
THE JOURNAL OF BIOWGICAL CHEMISTRY Vol. 258. No, 2, Issue of January 25, pp. 785-791, 1983 Prrnted in U.S A .
The Binding Propertiesof Human Complement ComponentC l q INTERACTION WITH MUCOPOLYSACCHARIDES* (Received for publication, April 5, 1982)
Sheri Almeda, RobertD. Rosenberg, and DavidH. Bing From the Center for Blood Research, Sidney Farber Cancer Institute, Beth Israel Hospital, and Harvard Medical School, Boston, Massachusetts 02115
Quantitative measurements have been made of the interaction of human complement subcomponent C l q with mucopolysaccharides.The bindingof C l q to heparin was quantitatively examined by utilizing an assay that employs a ‘‘%labeled low molecular weight heparin glycosaminoglycan (LMW-Hep) (Mr = 8500). Two classes of bindingsites were detected. The first class of l q with a Kd sites bound 2.02 mol of LMW-Hep/molCof of 76.6 ma. The second class of sites complexes with 12 molof LMW-Hep with a Kd of 1.01 pM. The higher affinity-bindingsite for LMW-Hep could be assigned to the collagenous region of Clq (Clq-c); 2.2 mol of 1251LMW-Hep were bound/mol of purified isolated Clq-c with a Kd = 381 nM. In contrast, the isolated clq globular region did not bind to ‘251-LMW-Hep. Thebinding of LMW-Hepto C l q and the Clq-c region was confirmed by fluorescence polarization experiments; C l q and Clq-c bound 2.3 and 2.02 mol of fluorescamine-labeled LMW-Hep/molof protein, respectively. A variety of mucopolysaccharides were able to inhibit interaction the most effective being of C l q with 1251-LMW-Hep, heparan sulfate and dermatan sulfate. LMW-Hep (2.5 11~) inhibited the ability of C l q (0.5 m) to recombine with ClF (1.4 m)and ClS (1.6m)to form hemolytically active Ci. At 250mM, LMW-Hepinhibited the hemolytic activity of reconstituted C i . The ability of mucopolysaccharides to interact with purified C l q suggests a role for such molecules in the regulation of the first component of complement.
It was noted 50 years ago that heparin-like materials exhibit anticomplementary activity (1).Numerous studies from i940 to the present have indicated that this inhibitory effect of the mucopolysaccharide is time-dependent as well as reversible. The anticomplementary activity of heparin is a function of the size of the complex sugar aswell as the content of carboxyl of the groups, but is independentof the anticoagulant activity mucopolysaccharide (2-5). The ability of heparin to inhibit the complement system is shared by a variety of other highly ~
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MATERIALS AND METHODS
Proteins Clq was purified as described by Bing et al. (22) and was further adsorbed withgelatin-Sepharose to remove anytraces of plasma fibronectin. Analysis on sodium dodecyl sulfate-7.5% polyacrylamide gels and by immunoelectrophoresis with anti-whole human serum, anti-ClF, anti-Cli,anti-IgG, and anti-IgMfailed to detect any contaminating proteins. Clq retained activity when combined with ClF and ClS ina functional assay which employs EAC4, C2, and CEDTA (22). The collagenous portion of Clq (Clq-c) was isolated from a pepsin hydrolysate of Clq by gel filtration on a Sephacryl 200 column (1.2 X 100 cm) as described by Reid (23).The globular region of Clq (Clqg) was prepared as outlined by Hughes-Jones and Gardner (24). C i was purified by affinity chromatography on IgG-Sepharose (25) modified to include elution at pH 7.4 with diaminopropane buffer (22).
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’ This work was supported by Grants AM 17351, HL 24856, ana HL 19131 from the National Institutesof Health, and Grant-in-aid82 947 from the American Heart Association. The costsof publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Complement terminology is that suggested by the Bull. W. H. 0. (1968) 39,935. Thus, Clq is a subcomponent of C1, the first component. C1F and CIS are the serine protease subcomponents of C1. C2 is the second component and C4 is the fourth component. CEDTA is the functional equivalent of terminal componentsC3-C9 prepared by diluting serum into 10 mM EDTA. An activated component is indicated by a bar across the component.
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charged components including dextran sulfate, carrageenan, and collagen (6-13). One site of action of mucopolysaccharides within the complement system has been shown to be the first component of this mechanism, Cl.’ Polyanionic polymers are able toinhibit the binding of activated C l t o EA2 or EAC4 by complexing with the Clq subcomponent (14, 15). Studies of the direct interaction of Clq with heparin and syntheticpolymers have indicated that there are a limited number (2 to 6) of binding sites on Clq,inhibition is competitive, and theKd is less than micromolar (16-19). However, the structural basisfor the interaction of Clq with polyanionic molecules such as heparin has yet tobe defined either in terms of the structural features of the protein (20, 21) or the polyanion (6). In this paperwe present data which show that there aretwo high affinity sites on purified Clq for a heparin glycosaminoglycan of M , 8500 and that theKc!of this interaction is 76.6 nM, and we provide evidence that thecollagenous portion of Clq contains bothof the above binding domains. Furthermore, we evaluate the ability of anticoagulantly active and nonanticoagulantly active forms of heparin aswell as othermucopolysaccharides to bind to theabove region of Clq. Based upon theavailable data, we hypothesize that heparin-like molecules may regulate the activity of complement via interactions with the first component of complement.
Mucopolysaccharides Porcine intestinal heparin with anticoagulant activity of 170 units/ mg (Sigma; M , = 15,000), whale cartilage chondroitin-4-sulfate ( M , = 25,000 to 56,000), shark cartilage chondroitin-6-sulfate ( M , = 40,000 to 80,000), porcine skin dermatan sulfate, and heparan sulfate were obtained as previously described (26). Porcine heparin was also purchased from Diosynth, The Netherlands, a t a n early stage in the ’The abbreviations used are: EA, the sheep erythrocyte-rabbit antibody complex used as the target in complement functional assays; LMW-Hep, low molecular weight ( M r= 8,500) heparin; Clq-c, theM , = 176,000collagenous portion of Clq; Clq-g, the M , = 39,000 globular portion of Clq.
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and Mucopolysaccharides
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manufacturing process prior to treatment with oxidizing agents and further purified by precipitation with cetylpyridinium chloride (27). The LMW-Hep of M , = 8,500 used in the radiobinding assay was isolated from the above material by fractionationon Sephadex G-100 (1.8 X 200 cm) equilibratedwith 150 mM NaCl in 10 mM Tris-HC1, pH 7.5. The molecular weight of this component was estimated by gel filtration chromatography utilizing a Sephadex G-I 00 column equilibrated with 500 mM NaCl in 10 mM Tris-HCI, pH 7.5, and calibrated with heparin standardsof known molecular size (27). Prior tolabeling with "'I, the LMW-Hep was concentrated by rotary evaporation and dialyzed against deionized water. Heparin species of high and low affinity for antithrombin 111 were obtained from the above mucopolysaccharide by affinity chromatography as described by Jordan etal. (27). Both components were rechromatographed on Sephadex G-100 as outlined above in order to obtain products with identical molecular size distributions. Measurement of Potential Mucopolysaccharide Concentration The levels of Clq, Clq-c, and Clq-g were determined either by absorbance measurementsa t 280 nm orby the modified Fohn method (28). The extinction coefficients of Clq and Clq-g were assumed to be 6.3 (23) and 7.0 (24). respectively.The molar concentrationof Clq, Clq-c, and Clq-gwere calculated utilizing molecular weights of 410,000 (20). 176,000 (25). and 39,000 (24),respectively. The levels of mucopolysaccharides were estimated colorimetrically by assay of uronic acid (29). The relationship between the above parameter and dry weight of mucopolysaccharide was determined experimentally. Radiolabeling of Heparin The LMW-Hep was labeled in two steps involving modification acid-N-hydroxysuccinimide ester with 3-(4-hydroxyphenyl)propionic followed by iodination with NaI2'I by a method which used chloramine-T as the catalyst (30, 31). Briefly, 3 mg of the LMW-heparin obtained by gel filtration was dialyzed against waterin boiled dialysis tubing (Spectrapore dialysis tubing, molecular weight cutoff
