rapid purification of the human c3b/c4b receptor (cr1) by monoclonal antibody affinity...

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Journal oflmmunological Methods, 82 (1985) 303-313 303 Elsevier JIM03604 Rapid Purification of the Human C3b/C4b Receptor (CR1) by Monoclonal Antibody Affinity Chromatography Winnie W. Wong l, Richard M. Jack ~, John A. Smith 2, Christine A. Kennedy 1 and Douglas T. Fearon ~'* i Brigham and Women's Hospital, Department of Rheumatology and Immunology, and Harvard Medical School, Department of Medicine, 607 Seeley G. Mudd Building, 250 Longwood Avenue, Boston, MA 02115, and 2 Massachusetts General Hospital, Departments of Molecular Biology and Pathology, and Harvard Medical School, Department of Pathology, 50 Blossom Street, Boston, MA 02114, U.S.A. (Received 18 April 1985. accepted 13 May 1985) The human C3b/C4b receptor (CR1) is a polymorphic glycoprotein that is expressed on erythrocytes, leukocytes and glomerular podocytes. Further structural analysis and molecular genetic studies would be facilitated by the availability of relatively larger amounts of purified CR1. Milligram quantities of CRI were purified from erythrocyte membranes 10,000-fold with an average yield of 30-40% by a rapid procedure which utilized sequential chromatography on Matrex Red A and a monoclonal anti-CR1 antibody affinity column. The purified receptor was homogeneous by SDS-PAGE and consisted of the 2 most common alleles of CR1. Purified CR1 also retained its function of serving as a cofactor for the cleavage of C3b to iC3b, C3dg and C3c. The amino acid composition was typical of that of a globular protein and sequence analysis of the N-terminus of the purified CR1 revealed that it was blocked. Key words: human CRI purification - C3 - monoclonal antibody - amino acid composition Introduction The purification of the human C3b/C4b receptor (CR1) has greatly facilitated studies of this membrane protein during the last 5 years. Not only was the receptor identified as a glycoprotein of M r 250,000 having regulatory activities analogous to those of factor H and C4 binding protein (Fearon, 1979; Iida and Nussenzweig, * To whom reprint requests should be addressed. Abbreviations: CR1, the human C3b/C4b receptor; EDTA, ethylenediamine tetraacetate; DFP, diiso- propylfluorophosphate; DOC, deoxycholic acid; I, C3b/C4b inactivator; NP-40, Nonidet P-40; PAGE, polyacrylamide gel electrophoresis; PBS, 10 mM sodium phosphate, 150 mM NaC1, pH 7.4; PBNS, PBS containing 1% NP-40 and 0.2% SDS; PMSF, phenylmethylsulfonylfluoride; Pth. 3-phenyl-2-thiohy- dantoin; SDS, sodium dodecyl sulfate. 0022-1759/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

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Journal oflmmunological Methods, 82 (1985) 303-313 303 Elsevier

JIM03604

Rapid Purification of the Human C3b/C4b Receptor (CR1) by Monoclonal Antibody Affinity

Chromatography

Winnie W. Wong l, Richard M. Jack ~, John A. Smith 2, Christine A. Kennedy 1 and Douglas T. Fearon ~'*

i Brigham and Women's Hospital, Department of Rheumatology and Immunology, and Harvard Medical School, Department of Medicine, 607 Seeley G. Mudd Building, 250 Longwood Avenue, Boston, MA 02115,

and 2 Massachusetts General Hospital, Departments of Molecular Biology and Pathology, and Harvard Medical School, Department of Pathology, 50 Blossom Street, Boston, MA 02114, U.S.A.

(Received 18 April 1985. accepted 13 May 1985)

The human C3b/C4b receptor (CR1) is a polymorphic glycoprotein that is expressed on erythrocytes, leukocytes and glomerular podocytes. Further structural analysis and molecular genetic studies would be facilitated by the availability of relatively larger amounts of purified CR1. Milligram quantities of CRI were purified from erythrocyte membranes 10,000-fold with an average yield of 30-40% by a rapid procedure which utilized sequential chromatography on Matrex Red A and a monoclonal anti-CR1 antibody affinity column. The purified receptor was homogeneous by SDS-PAGE and consisted of the 2 most common alleles of CR1. Purified CR1 also retained its function of serving as a cofactor for the cleavage of C3b to iC3b, C3dg and C3c. The amino acid composition was typical of that of a globular protein and sequence analysis of the N-terminus of the purified CR1 revealed that it was blocked.

Key words: human CRI purification - C3 - monoclonal antibody - amino acid composition

Introduction

T h e pu r i f i ca t ion of the h u m a n C 3 b / C 4 b receptor (CR1) has great ly fac i l i ta ted s tudies of this m e m b r a n e p ro te in d u r i n g the last 5 years. N o t o n l y was the receptor iden t i f i ed as a g lycopro te in of M r 250,000 h a v i n g r egu la to ry act ivi t ies a n a l o g o u s to those of factor H a n d C4 b i n d i n g p ro te in (Fea ron , 1979; I ida a n d Nussenzweig ,

* To whom reprint requests should be addressed. Abbreviations: CR1, the human C3b/C4b receptor; EDTA, ethylenediamine tetraacetate; DFP, diiso-

propylfluorophosphate; DOC, deoxycholic acid; I, C3b/C4b inactivator; NP-40, Nonidet P-40; PAGE, polyacrylamide gel electrophoresis; PBS, 10 mM sodium phosphate, 150 mM NaC1, pH 7.4; PBNS, PBS containing 1% NP-40 and 0.2% SDS; PMSF, phenylmethylsulfonylfluoride; Pth. 3-phenyl-2-thiohy- dantoin; SDS, sodium dodecyl sulfate.

0022-1759/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

304

1981; Medof et al., 1982; Ross et al., 1982), but specific polyclonal and monoclonal antibodies could be prepared for the first time (Fearon, 1980; lida et al., 1982). These reagents were employed to enumerate receptors on the various cell types having CR1 (Fearon, 1980; Iida et al., 1982; Wilson et al., 1982, 1983). Chemotactic peptides and phorbol diesters were found to regulate the expression of CR1 on the surface of myelomonocytic cells (Fearon and Collins, 1983; Berger et al., 1984: Lee et al., 1984; Changelian et al., 1985), and erythrocytes were shown to exhibit genetically determined differences in CR1 expression (Wilson et al., 1982). De- ficiencies of this receptor were found on several cell types in patients with autoim- mune or infectious diseases (reviewed in Wilson and Fearon, 1984). Reciprocal co-capping of CR1 and Fc receptors on neutrophils was demonstrated and internali- zation of CR1 that had been crosslinked with antibody was shown to occur through coated pits (Fearon et al., 1981; Abrahamson and Fearon, 1983; Jack and Fearon, 1984). A role for receptor in activation of B cells was suggested (Daha et al., 1984) and a soluble form of CR1 which resembled membrane CR1 in size and in cofactor activity for the cleavage of C3b and iC3b has been found in human plasma (Yoon and Fearon, 1985). In addition to these functional studies of CR1, structural and biosynthetic studies of the receptor were initiated, revealing a genetically regulated polymorphism of size and the presence of N-linked oligosaccharides (Dykman et al., 1983, 1984, 1985; Wong et al., 1983; Atkinson and Jones, 1984). The definition of the structural polymorphism made possible the demonstration of genetic linkage of CR1 with C4 binding protein (De Cordoba et al., 1984).

In order to investigate more comprehensively the structure of CR1, however, relatively larger quantities of purified protein will be required than have been necessary for previous studies. Although a ready source of CR1 is available in the erythrocyte, shortcomings of published methods for the purification of the receptor have been low yields and multiple chromatographic steps. In the present study, a rapid 2-step procedure using a monoclonal anti-CRl affinity column is described that leads to a 10,000-fold purification of functional CR1 from human erythrocyte membranes with a 30-40% yield.

Materials and Methods

Preparation of erythrocyte membranes Eight units of outdated whole blood were obtained from the Red Cross. Plasma

and leukocytes were removed by 3 washes in 5 vols. of ice-cold phosphate-buffered saline (PBS, 10 mM sodium phosphate, 150 mM NaCI, pH 7.4) containing 2 mM disodium ethylenediamine tetraacetate (EDTA), 100 ~M phenylmethyl- sulfonylfluoride (PMSF) (Sigma, St. Louis, IL), 1 /~M each of leupeptin, pepstatin and 0.02% NaN 3. The erythrocytes were lysed in 6 vols. of ice-cold 10 mM Tris, pH 8.0, containing 1 mM EDTA, PMSF, leupeptin, pepstatin, and NaN 3 as above. Concentration of the membranes and hemoglobin removal were achieved by con- tinuous washing with the same buffer in a Pellicon cassette system (Millipore, Bedford, MA) that had been fitted with 2 cassettes containing Durapore 0.5 /~m filters (Millipore). The hemoglobin-free ghosts were stored at - 7 0 ° C until use.

305

Chromatography media Matrex Red A (Amicon, Danvers, MA) was stored in 10 mM Tris containing 500

mM KC1, 2 mM EDTA and 0.02% NaN 3, pH 8.0. The gel was regenerated after each use or after prolonged storage by washing with 10 vols. of 8 M urea and 0.5 M NaOH.

Nonimmune rabbit IgG and YZ-1 IgG 1, a murine monoclonal anti-CR1 (Chan- gelian et al., 1985), were coupled to CNBr-activated Sepharose (Sigma) at densities of 8 and 5 mg/ml, respectively. The Sepharose-YZ-1 was prewashed with the CRl-eluting buffer of 150 mM triethanolamine, pH 12.2, containing 500 mM KC1 and 0.5% DOC. Purified CR1 was labeled with 125I-Bolton-Hunter reagent (ICN Pharmaceutical, Irvine, CA) (Wong et al., 1983) and used as a tracer during purification of CR1.

Radioimmunoassay CR1 content was measured by a modification (Yoon and Fearon, 1985) of the

sandwich radioimmunoassay of Unkeless and Healy (1983). The YZ-1 monoclonal anti-CR1, an IgG 1 immunoglobulin that does not bind to protein A, was labeled with ~25I to a specific activity of 1-2 × 106 cpm//~g (Fraker and Speck, 1978). Replicate samples of 160 ng 12SI-YZ-1 IgG in 80 ~1 of PBS containing 1% Nonidet P-40 (NP-40) (Gallard-Schlesinger, Carle Place, NY), 0.2% sodium dodecyl sulfate (SDS) (Bio-Rad, Richmond, CA), 2% heat-inactivated fetal bovine serum, 5 mM diisopropylflourophosphate (DFP) (Sigma), 1/~M leupeptin (Sigma), and 0.1% NaN 3 were incubated for 60 min at 4°C with 20/xl of buffer or of fractions to be assayed for the presence of CR1. Twenty/~1 of a 10% suspension of formalin-fixed Staphylo- coccus aureus particles (Bethesda Research Laboratories, Bethesda, MD) that had been precharged with rabbit IgG anti-CR1 were added and incubation was con- tinued for 60 min at 4°C. The S. aureus particles were washed 3 times with 0.5 ml of the reaction buffer and the pellets were assessed for the amount of specifically bound 125I-YZ-1-CR1 complexes.

Cofactor activity of CR1 C3 (Hammer et al., 1981), factor H (Weiler et al., 1976), and factor I (Fearon,

1977) were purified as described. C3 was converted to C3b by incubation for 1 min at 37°C with 0.2% z-l-tosylamide-2-phenylethylchloromethyl ketone (TPCK)-treated trypsin (Cooper Biomedical, Malvern, PA) followed by addition of 5 mM DFP. The C3b was radiolabeled to a specific activity of 3 × 105 cpm//tg (Fraker and Speck, 1978). In order to compare the relative cofactor activities of purified and nonpuri- fied CR1, 125I-C3b at 5.7 #g/ml and factor I at 27 #g/ml were incubated with 267 /~g/ml of affinity-purified rabbit IgG anti-CR1 or nonimmune rabbit IgG, and incremental amounts of purified CR1 or freshly solubilized crude CR1 from erythrocyte membranes for 60 min at 37°C in PBS containing 1% NP-40, 0.2% SDS (PBNS), 5 mM DFP, 2 mM EDTA, 1 mM PMSF and 1 #M leupeptin. The reactions were stopped by the addition to each tube of an equal volume of 125 mM Tris with 6% SDS, 8 M urea, 200 mM dithiothreitol, 0.2% bromophenol blue and 10% glycerol, pH 6.8. After boiling for 3 min at 100°C, the samples were analyzed by

306

SDS-PAGE (Laemmli, 1970) on slabs containing a gradient of 5-15% acrylamide and autoradiography. The regions of the dried gel corresponding to the c~'- and B-chains were cut out and assessed for 12sI in a Searle Model 1185 gamma counter (Searle, Chicago, IL).

In order to analyze the cleavage of C3b to C3c and C3dg, 125I-C3b at 19.5 btg/ml and factor I at 91 /ag/ml were incubated with factor H at 364 /~g/ml or with incremental amounts of purified CR1 in PBNS with 5 mM DFP, 2 mM EDTA, 1 mM PMSF, and 1 /xM leupeptin. After incubation for 60 rain at 37°C, the reactions were stopped as above and the products were analyzed by SDS-PAGE.

Amino acid analysis Amino acid compositions were determined using a Beckman 6300 analyzer

equipped with 2 Hewlett-Packard 3390A integrators. The samples were hydrolyzed in sealed, evacuated, 6 × 50 mm Pyrex tubes at l l0°C for 24 h or 48 h in 25 ill of constantly boiling HC1 (Sequanal Grade, Pierce Chemical, Rockford, IL) (Moore, 1972). Ninhydrin and 2-channel (440 and 570 nm) integration provided analysis of all amino acids, except tryptophan, cysteine, asparagine and glutamine, with a lower limit of detection of 25 pmol and with confidence values of 1-8% at 100 pmol/amino acid. Analyses were done 3 times on each of 2 separately purified samples of CR1 containing approximately 60 pmol protein and background controls.

Protein sequence analysis Automated Edman degradation was performed with an Applied Biosystems 470A

sequencer. The sequence program was developed earlier for the gas-liquid solid-phase peptide and protein sequencer (Hewick et al., 1981). The no vacuum program contains one coupling step at 44°C for 32.1 rain and a single cleavage step at 44°C for 15.0 rain. Automated conversion of the 2-anilino-5-thiazolinone derivatives into the corresponding 3-phenyl-2-thiohydantoin derivatives (Pth-amino acids) was achieved in 25% trifluoroacetic acid for 32.3 min at 50°C. Polybrene was added to the glass filter disk in the cartridge prior to degradation of protein (Klapper et al., 1978; Tart et al., 1978), and 5 sequence cycles were run to reduce Polybrene-related contaminants. All Pth-amino acids, as well as S-carboxymethyl cysteine and c-suc- cinyl lysine were identified by high performance liquid chromatography on 0.45 × 25 cm analytical cyanopropyl columns (IBM, Danbury, CT) using 15 mM sodium acetate, pH 5.8, which contained 5% tetrahydrofuran and a linear gradient of acetonitrile based on 'a system developed by Hunkapiller and Hood (1983) and modified by W. Touchstone (unpublished data). This HPLC system separates the Pth-amino acids and internal standard (methyl ester of Pth-Glu) from the major contaminants: a dithiothreitol adduct, a dimethylamine/phenylisothiocyanate ad- duct and diphenylthiourea. Two hundred pmol of an internal standard were added to each cycle collection, and the samples were dried in a Speed Vac Concentrator with a RH 100-6 rotor (Savant, Hicksville, NY). The dried samples were dissolved separately in 25/~1 of water/acetonitrile (2 : 1, v/v) and a 17 ~tl aliquot representing 68% of each sample was injected automatically with a WISP 710B (Waters, Milford, MA). The Pth-amino acids were detected by UV absorbance at 254 nm and 313 nm

307

using 2 Beckman 160 detectors which were interfaced with a Nelson Analytical (Cupertino, CA) Model 3000W chromatography system and an IBM PC-XT. The IBC PC-XT was further interfaced to a VAX 11/780 (Digital Equipment Corpora- tion, Maynard, MA).

Results

All procedures were carried out at 4°C except the elution of CR1 from Sepharose- YZ-1 monoclonal anti-CR1 which was performed at 20°C. In a typical purification, washed membranes from 5.9 x 1013 erythrocytes were suspended at a concentration of 1.6 x 101° ghosts/ml in a final volume of 3.7 liters of 10 mM Tris-HCl, 50 mM KCI, pH 8.1, containing the detergents, 0.2% NP-40 and 0.3% DOC, and the inhibitors, 5 mM DFP, 200/~M PMSF, 2 mM EDTA, 2 mM iodoacetamide, 5/~M leupeptin, 1 /~M pepstatin and 0.02% NaN 3. In the initial description of the purification of this membrane protein (Fearon, 1979), NP-40 had been found to be sufficient for solubilizing CR1 from erythrocyte membranes when the buffer con- tained an isotonic concentration of NaCI. In the present study, however, the hypotonic salt concentration that was chosen to enhance the subsequent binding of CR1 to Matrex Red A necessitated the addition of DOC to the buffer to achieve greater than 90% solubilization of the receptor.

Without removing detergent-insoluble material, the lysate of the erythrocyte membranes was added directly to a moist cake of 700 ml of Matrex Red A that had been equilibrated in the solubilizing buffer. The Matrex Red A was suspended, intermittently stirred for 2 h and incubated overnight at 4°C. The mixture was poured into a coarse sintered glass funnel which retained the Matrex Red A but through which detergent-insoluble and other unbound materials could pass. The gel was washed with 10 vols. of solubilizing buffer and then poured into a 5 cm X 50 cm column, packed, and washed with 1 vol. of solubilizing buffer. The absorbed proteins were eluted with this buffer containing an additional 0.2% DOC and 1.45 M KC1 and the inhibitors, 5 mM DFP, 1 mM PMSF, 2 mM EDTA, 2 mM iodoaceta- mide, 5/~M leupeptin, 1/~M pepstatin and 0.02% NaN 3. CR1 and protein coeluted and the 200 ml pool of CRl-containing fractions had 72.5% of the receptor but only 2.4% of the protein that had been present in the starting material (Table I). In 2 other preparations of receptor, the yields of CR1 at this stage were 75% and 84%, and the purification factors were 29- and 30-fold, respectively.

The pool of eluted proteins was concentrated to 80 ml by high pressure ultrafiltra- tion through an XM-100 Diaflo membrane (Amicon) in order to decrease the volume to be applied to the affinity column of Sepharose-YZ-1 anti-CR1. Despite no CR1 being detectable in the filtrate, only 63% of the receptor was recovered in the retentate. In 2 other preparations the recoveries of CR1 were 69% and 87% and this loss may reflect binding of CR1 to the filter. The concentrated pool was dialyzed overnight against 6 liters of PBS containing 0.5% NP-40, 2 mM EDTA, 1 mM PMSF, 1 mM iodoacetamide and 0.02% NaN 3. After dialysis the concentrate was pumped through a preclearance column of 7 ml of Sepharose-nonimmune rabbit

308

TABLE I

PURIFICATION OF CR1

Total Total Total volume protein a CR1 h (ml) (mg) (rag)

Washed E from 29 U of blood 7400 Hemoglobin-free ghosts 3 300 Detergent solubilized ghosts 3 700 Fall through of Matrex Red A 3 000 Pool of Matrex Red A Eluate 220 XM-100 filtrate 144 Starting material for Sepharose-YZ-1

(concentrated and dialyzed pool) 78 Eluate of Sepharose-YZ-1

(concentrated and dialyzed) 3.5

37000 10.2 26 300 0.38

880 7.4 0 0

800 4.7

1.47 3.68

Protein was determined by the method of Lowry (1951) employing bovine serum albumin as standard. h CR1 wasquantified by radioimmunoassay as described in Materials and Methods.

IgG that was connected in tandem to a 2 ml affinity column of Sepharose-YZ-1. The flow rate was maintained at 3 -4 m l / h because faster flow rates were associated with CR1 appearing in the fall-through. When all the material had been loaded, the preclearance column was disconnected and the Sepharose-YZ-l-column was sub- jected to the following washes at a flow rate of 20 ml /h ; 5 vols. of PBNS; 5 vols. of 100 mM sodium acetate with 150 mM NaC1 and 0.5% NP-40, pH 4.5; 10 vols. of PBNS; 10 vols. of 10 mM Tris-buffered saline containing 0.2% SDS and 0.25% DOC, pH 9; and 10 vols. of 150 mM triethanolamine with 500 mM KC1 and 0.5% DOC, pH 10 (Fig. 1). CR1 was then eluted at room temperature with the tri- ethanolamine buffer adjusted to pH 12.2, and fractions of 1 ml were collected and immediately neutralized by the addition of 30/~1 of I M HC1. All buffers used in the above procedure contained the following inhibitors: 1 mM PMSF, 2 mM EDTA, 2 mM iodoacetamide and 0.02% NaN 3. Immediately after elution the column was washed with PBNS and stored in PBS with 1 mM PMSF, 2 mM EDTA and 0.02% N a N 3 at 4°C. This affinity column could be reused at least 4 times. The purified CR1 was dialyzed twice against 1000 vols. of 100 mM N H 4 H C O 3 with 0.05% Zwittergent 3-14 (Catbiochem-Behring, San Diego, CA), concentrated in a Savant Speed Vac Concentrator and stored at - 7 0 ° C . This affinity purification step effected a 78% recovery and a 426-fold purification of CR1 (Table I); in 2 other preparations the yields of CR1 were 65% and 70% and the purification factors were 277- and 321-fold. The final yield of CR1 averaged 39% of the receptor present in the washed erythrocyte membranes and the purification factor ranged from 8310 to 13,240 for 3 preparations. On SDS-PAGE, the purified receptor demonstrated 2 Coomassie blue stained bands corresponding to the most commonly occurring allotypic forms of this glycoprotein (Fig. 2).

The amino acid composition was determined for 3 replicate samples of 2

309

1 2

,,p

I

pH 4.5 I,

16

t2

8

4

13

pH 7.4 pH 9.0 pl.I t0.0 pH t22

' ' i i I I 6 ,b "A ~o 4o 50 60 ro FRACTION NUMBER

9 5 -

6 8 -

4 3 -

3 0 -

Fig. 1. The elution of CR1 from Sepharose-YZ-I with buffers of differing pH. Partially purified CR1 derived from the Matrex Red A column and containing a small amount of 1251-labeled purified CR1 was applied to a Sepharose-YZ-1 affinity column. The column was washed and eluted with buffers having the indicated pH values, and 125I content of the fractions was assessed.

Fig. 2. Analysis of purified CR-1 by SDS-PAGE followed by staining with Coomassie blue; lanes 1 and 2, 7.5 #g and 15/~g, respectively, of unreduced, purified CR1.

preparations of purified CR1. There were no unusual amino acids detected (Table II). Amino terminal analysis of 1.46 nmol of CR1 led to the detection of less than 100 pmoles of any amino acid in the first 2 cycles. This loW"yjeld indicates that the amino terminus of CR1 is blocked and further suggests that contaminants comprise at most 7% of the total protein in the CR1 preparation, assuming that putative contaminants are not blocked at their amino termini.

The capacity of purified CR1 to serve as a cofactor for the factor I-mediated cleavage of ]25I-C3b was compared to that of CR1 present in the detergent extracts of human erythrocyte membranes. Replicate samples of t25I-C3b were incubated for 60 min at 37°C with factor I, non-immune rabbit IgG or affinity-purified IgG anti-CR1 and 0-50 /~g/ml of purified CR1 or 0-0.5 /~/ml of CR1 as found in extracts of erythrocyte membranes. The ]25I-C3b was then analyzed by SDS-PAGE and autoradiography. The percent of the a'-chain that had been cleaved was

100( --. -o l , , .~f~. . .~----c~---a,--~-.-o---~ . . . . .-o pCR1 + aCRI

80 " ~ * a c R t

. 60 ~Jl" cCR'+NIIgG~ ~

40 " ~ /pCRt+ NI IgG

20

, , __ / / n I n nn l an l n a J I J l Ln l , t J I , , t , I , , , I L , , ,

0 0.1 0.5 t 5 t0 50 ~00 [CRf], /zg/ml

Fig. 3. Dose-response analysis of the cofactor function of purified CR1 (pCR1) and crude CR1 solubilized from erythrocyte membranes (cCR1) for the cleavage of the a'-polypeptide of C3b by factor I. 125f-labeled C3b was incubated with factor I and incremental concentrations of pCR1 or cCR1 that had been pretreated with nonimmune (NI) IgG or rabbit IgG anti-CR1 (aCR1). After reduction with dithiothreitol, the labeled products of this reaction were subjected to SDS-PAGE and regions of the gel corresponding to the positions of the intact a'- and B-chains were excised and assessed for 125I in order to determine the percent residual intact a'-chain.

M r

200--

9 3 ~

6 8 ~

~i ~ ~ i i

Fig. 4. Cleavage of C3b to iC3b, C3c and C3dg by factor I and CR1 as assessed by SDS-PAGE and autoradiography. 125I-labeled C3b was incubated with factor I in the presence of 364/ tg /ml of factor H (lane 1), or in the presence of various concentrations of purified CR1 : 273 ~g/mt (lane 2); 137/*g/ml (lane 3); 69 #g /ml (lane 4); 35/~g/ml (lane 5); 14/~g/ml (lane 6); 7/~g/ml (lane 7); 3.5/~g/ml (lane 8); 0 /~g/ml (lane 9). 125I-C3b was also treated with 273 #g /ml of CR1 in the absence of factor I (lane 10).

T A B L E II

A M I N O A C I D C O M P O S I T I O N O F CR1

Amino acid Residues/100 residues d

311

Asx a 10.6

Thr b 5.5

Ser b 8.9

Glx a 9.5

Gly 9.1

Ala 4.7

Cys N.D. c

Val 7.1

Met 1.3

lie 4.8

Leu 7.4 Tyr b 2.8

Phe 5.0

His 2.9

Lys 4.1

Arg 5.1

Pro 11.2 T rp N.D. c

a Asx = Asp + A s n , and Glx = Glu + Gin.

b Corrected values. c N .D . = not determined (Cys and Trp are destroyed by acid hydrolysis). d Numbers represent the average of 3 determinations of 2 separate preparations of CR1. Confidence

,~alues are 1-2%.

determined by normalizing to a constant amount of fl-chain after the assessment of ~25I in the respective regions. Fifty percent of C3b was cleaved by factor I in the presence of 1.15 ~g /ml of purified CR1 and 340 ng/ml of crude CR1 in erythrocyte lysate (Fig. 3). Thus, the purified CR1 retained about 30% of the function of native CR1 in this assay.

The capacity of purified CR1, in its detergent soluble form, to induce the cleavage of C3b to C3c and C3dg factor I was examined by incubating tESI-C3b with factor I and 3.5-273/~g/ml of purified CR1 for 60 min at 37°C. The products were analyzed by SDS-PAGE and autoradiography. Conversion of all C3b to C3dg and C3c occurred with the 2 highest concentrations of purified CR1 (Fig. 4). Therefore, this unique function of CR1 was retained through the process of purification of the receptor.

Discussion

The structural characterization of the plasma protein components of the comple- ment system has been greatly accelerated by the application of molecular biological

312

techniques. Most of these studies, however, have been dependent on the availability of some amino acid sequences which served to direct the synthesis of oligonucleotide probes and to verify the nucleotide sequences of cloned cDNA. To carry out comparable studies of CR1, therefore, some knowledge of its primary structure is required and this, in turn, is dependent on the availability of purified receptor in amounts that are sufficient for amino acid sequence analysis. Although the erythro- cyte is a relatively convenient source of CR1, the density of CR1 on these cells is in the range of only 550 sites per cell and one liter of packed erythrocytes contains only 10 nmol of receptor. Therefore, the development of an efficient isolation scheme providing a high yield of purified CR1 was considered an essential step in prepara- tion for molecular biological studies of CR1.

The initial step in the purification of CR1 from the erythrocyte membrane lysate involved the use of batch-wise absorption to and elution from Matrex Red A (Table I). This procedure accomplished the aims of rapidly decreasing the protein content and volume of the detergent lysate without incurring a significant loss of CR1. Although the further concentration of CR1 by ultrafiltration was consistently associated with the loss of some receptor, we considered this loss to be preferable to the excessive length of time that would have been required to apply the noncon- centrated pool to the immunoaffinity column. Alternatively, several affinity columns can be run in parallel to save time. The most important step in the purification of CR1 was the use of Sepharose-YZ-1 which led to a 300- to 400-fold purification and a 60-80% recovery of CR1. The critical characteristic of the monoclonal antibody that made it suitable for this purpose was its relatively high affinity for CR1 (K d of 2 x 10 -9 M at pH 7.4) that accounted for the efficient uptake and retention of CR1 by the affinity column during extensive washing with buffers encompassing a wide pH range and containing the ionic detergent, SDS (Fig. 1). YZ-1 also effectively bound both major allotypic forms of CR1 (Fig. 2), indicating that it can be used for the purification of each form from homozygous donors. A disadvantage of the high affinity of the YZ-1 for CR1 was the requirement for a severely alkaline buffer to elute the antigen, since the exposure to pH 12.2 may have caused the diminished specific functional activity of the purified receptor relative to that of crude CR1 in lysates of erythrocyte membranes (Fig. 3). However, it is also possible that CR1 purified by more standard methods that require multiple chromatographic steps might exhibit a similar decrease in its functional capacity. Furthermore, purified CR1 retained its unique function of promoting the cleavage of iC3b to C3dg by factor I (Fig. 4).

The N-terminal sequence could not be obtained because the N-terminus of CR1 was blocked. The amino acid composition of CR1 was obtained and no unusual amino acids were detected (Table II). However, comparison of the composition of CR1 to those of factor H (Sim and DiScipio, 1982) and C4 binding protein (Reid and Gagnon, 1982) by the method of Marchalonis and Weltman (1971) gave SAQ values of 35 and 42, respectively. As comparisons of unrelated proteins showed only 2% to possess SAQ values within 100 U of each other, these values for the comparisons of CR1 to factor H and C4 binding protein are therefore significant and support other findings of the relatedness of these proteins.

313

Acknowledgments

This work has been supported in part by NIH Grants AI-10356, AI-19397, AI-22531, AI-22833, AM-20580, AM-35907, and RR-05669. W.W. is Postdoctoral Fellow of the Arthritis Foundation. J.A.S. is supported by a grant from Hoechst-AG (F.R.G.).

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