Functional consequences of the genetic polymorphism of the third component of complement

III

i

Functional consequences of the genetic polymorphism of the third component of complement Thomas R. Welch, MD, Linda Beischel, a n d A m y K l e e s a t t e l From the Department of Pediatrics, Universityof Cincinnati College of Medicine, and the Children's Hospital Research Foundation, Cincinnati, Ohio

The third component of complement, the central protein of the complement cascade, occurs in two principal allotypes, C3S and C3F. An excess frequency of the F aUotype has been implicated in a number of disease states, including some forms of glomerulonephritis. These associations have been explained by functional differences between C3S and C3F. We examined several complement functions, using purified preparations of C3S or C3F. The C3S allotype was 1.3 times more efficient than C3F in a hemolytic assay employing sensitized sheep erythrocytes; this difference was shown to arise from a slightly more efficient deposition of C3F on the cell surface. These differences are trivial and of much less magnitude than the functional differences between C4A and C4B. There were no differences between allotypes in their ability to be converted to inactive C3b (C3bi) by complement factors H and I or by CR1 and factor i. No significant differences were seen between the allotypes and their ability to support solubilization of preformed immune complexes. (J PEDIATR1990;116:$92-7)

The third component of complement, a three-chain polypeptide of Mr 190,000, is the central effector molecule of the complement cascade. As such, the protein plays an important role in host defense. In 1968, Alper and Propp 1reported that C3 displayed electrophoretic polymorphism. The two common allotypes, C3*F and C3"S, were detected, as well as a number of rare variants. These workers were unable to detect any difference in C3 hemolytic function in sera homozygous for one or the other of the alleles. 1 In 1974, Arvilommi2 examined the formation by mononuclear cells of rosettes with erythrocytes coated with either C3S or C3F. His experiments suggested that the C3F phenotype had a greater affinity for the monocyte C3 receptor than did C3S. Before and after Arvilommi's report, a n u m -

Reprint requests: Thomas R. Welch, MD, Divisionof Nephrology, Children's Hospital Research Foundation, Elland and Bethesda Avenues, Cincinnati, OH 45229-2899. 9/0/20151

$92

ber of epidemiologic studies addressed the distribution of C3 phenotypes in populations with disease. 3-s Most of these studies have yielded results of borderline significance, and some have subsequently been called into question.9, 10 Nonetheless, Arvilommi's study2 is frequently cited as BSA DEAE DGVB EDTA PAGE PBS PNHS RR SDS

Bovine serum albumin 2-Diethylaminoethanol Dextrose, gelatin, veronal buffer Ethylenediaminetetraacetic acid Polyacrylamidegel electrophoresis Phosphate-buffered saline solution Pooled normal human serum Relative risk Sodium dodecyl sulfate

showing a functional basis for distortions in C3 phenotype distribution in disease. In the 15 years that have followed this initial report, much has been learned about C3 function, the regulation of activated C3 (C3b), and complement receptors. Nonetheless, there have been no further detailed

Volume 116 Number 5

examinations of functional differences between the C3 phenotypes. This situation is in marked contrast to that of C4, in which detailed investigation of the functional consequences of isotypic differences have been reported. The C4 isotypic differences have also been consistently linked to disease susceptibility. It would seem that C3, by its central role, would be an even more promising component to demonstrate functional consequences of inherited polymorphism. Therefore we performed a detaited study of the relationship between C3 phenotype and serum protein concentration, hemolytic efficiency, control by regulators, and ability to solubilize preformed immune complexes; METHODS

Materials, reagents, and buffers. Sheep erythrocytes (Colorado Serum Co., Denver, Colo.), rabbit antisheep cells (7s) and complement components C5 to C9 (Diamedix Corp., Miami, Fla.), and bovine serum albumin (Sigma Chemical Co., St. Louis, Mo.) were purchased as described. Saline solution with dextrose-gelatin-veronal buffer contains 2.5% dextrose and 0.1% gelatin. DGVB § and PBS ++ contain 0.15 mmol/L calcium chloride and 0.5 m m o l / L magnesium chloride. Pooled normal human serum was depleted of functional C3 and C4 by being brought to 100 mmol/L methylamine for 30 minutes at 30 ~ C; methylaminc was then removed by passage over a Sephadex G-25 column (Pharmacia Inc., Piscataway, N.J.). (Monoclonal anti-C3c was a gift of Gordon Ross, P h D ) C3 eleetrophoresis. Immunofixation electrophoresis of serum was performed by a modification of the method of Atper and Propp, 1 as recently described from our laboratory.9 C3 serum eoneentrations. The serum concentration of C3 was measured by radial immunodiffusion, with the use of goat antisera. Measurements were made in the sera of 42 healthy, unrelated hospital personnel in whom C3 phenotypes were known. Complement component purification. C3 was purified from units of fresh citrated plasma from donors previously determined by agarose electrophoresis to be homozygous for either C3S or C3F. The plasma was initially absorbed batchwise with QAE-Sephadex A-50 beads (Pharmacia) according to the method of Lundwall et aL, tt so that both C3 and C4 could be obtained from the same material. C,4, and approximately 50% of the initial C3, were eluted in 0.5 mol/L sodium chloride after the QAE-Sephadex had been washed with 0.2 mol/L s6dium chloride and pelleted. This eluate was dialyzed and applied to a column (4.5 X 8.5 cm) of DEAE-Sephacel beads (Pharmacia). Separate peaks of C3 and C4 were eluted by a gradient to 0.3 mol/L sodium chloride, and the C3 pool was concentrated by precipitation

Functional consequences o f polymorphism

S93

with 16% (wt/vol) polyethylene glycol 4000 (Sigma). a2 Trace contaminants were removed by passage over a Sepharose 4B column (Pharmacia) to which goat antisera to C5, factor H, C4, IgG, IgA, and IgM had been conjugated. The final C3S and C3F preparations were concentrated and dialyzed into 0.5 mol/L sodium phosphate, pH 7.4, and 0.15 mol/L sodium chloride in a Micro-ProDiCon protein concentrator (Bio-Molecular Dynamics, Beaverton, Ore.) and stored in a!iquots at - 8 0 ~ C. The C3 preparations were shown to be free of contaminating proteins by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE technique). In addition, 5 ug of each preparation was incubated with complement factors H (5 #g) and I (0.25 #g) for 30 minutes at 37 ~ C. No proteolytic conversion to C3bi was shown by SDS-PAGE of these preparations, implying that the thiol ester bond was not disrupted during isolation. Complement factor H was obtained as a by-product of C3 isolation. Fractions from the DEAE,Sephacel column that contained factor H were pooled, dialyzed, and passed over D N A cellulose (Sigma). The retained factor H was eluted by 0.5 mol/L sodium chloride) 3 Complement factor I was functionally purified from normal serum containing 0.04 mol/L EDTA by immunoabsorption and elution from Sepharose 4B anti-I. 14 Functionally pure C1 was prepared as reported, is C3 hemolytic efficiency. The specific functional activities of the purified C3S and C3F preparations were evaluated by a stoichiometric hemolytic titration. Sheep erythrocytes were optimally sensitized with rabbit antibody. These erythrocyte antibodies (EA) were reacted sequentially 16 with 500 effective molecules per cell of partially purified C1, with 350 molecules per cell of purified C4, and again with C1. These EAC14 complexes, 1 • 107 per reaction tube, were mixed with oxidized C2,17 an appropriate dilution of C3, and a mixture of C5, C6, and C7. After a 30-minute incubation at 30 ~ C, C8 and C9 were added and the mixture was incubated at 37 ~ C for 60 minutes. The buffer used for this assay was DGVB ++. The reaction mixtures were centrifuged, and the optical density of the hemoglobin released into the supernatant was determined by its absorption at 413 nm in a Zeiss model PMQII spectrophotometer. The dilution of the titrated component that lyses 63% of the sensitized cells is considered to provide one effective molecule per cell C3S, C3F, and a pooled normal serum of known C3 concentration were always assayed simultaneously and the effective molecules per microgram (hemolytic efficiency) of C3S and C3F expressed as a percentage of that obtained for the pooled serum. Interaction of C3 with factor H or CRI. Purified C3S or C3F was converted to C3b by incubation for 5 minutes with 0.22% (wt/wt) L-l-p-tosylamino-2-phenylethyl chloro-

S 94

Welch, Beischel, and Kleesattel

The Journal of Pediatrics May 1990

Toble I. Differential binding of C3S and C3F to erythrocytes C3 added (#g/t07 cells) 0.2 0.4 0.8 1.6 3.2

Molecules of anti-C3 bound/cell C35

C3F

Ratio S/F"

1.5 • 10 4 2.8 • 104 5.2 x 10 4 8.5 • 10 4 1.6 • 10 4

1.2 • 104 2.0 • 104 3.6 X 10 4 6.2 X 10 4 1:2 • 10 4

1.25 1.40 1.44 1.37 1.33

Uptakeof C3 ontocellswasdeterminedafterincubationofC3F or C3S with EAC!4 and C2. SurfaceC3b was quantitatedby the bindingof 125Imonoclonalanti-C3c.Data are from a representativeexperiment. *Mean ratio: 1.3&

Table I1o Solubilization of preformed immune complexes by C3S and C3F % Solubillzation Time (mln)

C3S

C3F

30 35 34 120 63 66 BSA-anti-BSAcomplexeslabeled with 125Iwere incubatedwith methylamine-treatedPNHS reconstitutedwith C3S and C3F. PercentsolubilizatJonof the complexeswas assessedat timedintervalsby releaseof radioactivityintothe supernatant,Eachvaluerepresentsthe meanof three separate experiments.

methyl ketone (TPCK) trypsin (Sigma), followed by a 5.-minute incubation with 0.44% soybean trypsin inhibitor (Sigma). C3b(F) or C3b(S), 2 #g, was incubated for 30 minutes at 37 ~ C with 0.1 gg factor I and increments of factor H in a constant total volume of PBS. C3b (allotype F or S) was also incubated with factor I and Various volumes of pelletedl washed human erythrocytes, a source of the C3 b receptor CR 1.18 Erythrocyte concentration was determined on a Coulter Counter automated cell counter (Diff 350, Coulter Electronics, Hialeah, Fla.), and leukocyte contamination was less than 0.1%. All C3b mixtures were reduced with 2-mercaptoethanol (Bio-Rad Laboratories, Chemical Division, Richmond, Calif.), boiled for 3 minutes with SDS, and electrophoresed on an 8% polyacrylamide gel slab 19 in a Protean Cell apparatus (Bio-Rad). A Quik Scan densitometer with peak integration by the Quik Quant II peak in-" tegrator (Helena Laboratories, Beaumont, Tex.) was used to determine the relative intensities of the stained (Coomassic Blue) protein bands. Immune complex solubilization, BSA-anti-BSA immune complexes labeled with iodine 125 were made at equiva-

lence, as previously described. 2~ Methylamine-treated PNHS was reconstituted with 1.5 mg/ml C3S or C3F. Immune complexes (1/~g BSA) were added to 1.5 ml of the reconstituted PNHS, diluted 1:1 with PBS ++. Timed aliquots were removed and centrifuged, and the percentage of total radioactivity in the supernatant was used as a measure of the percentage of immune complexes solubilized. Uptake of solubilized complexes. Human erythrocytes, 1 X 108 in 0.15 ml PBS ++, were preincubated with 0.15 ml of a 1:40 dilution of PNHS in PBS ++ and 0.15 ml of a 1:10 dilution of C3F or C3S in PBS ++ for 10 minutes at 37 ~ C in multiple microcentrifuge tubes. Suspended BSA-antiBSA complexes (containing 1 #g BSA), labeled with iodine 125, were added to each tube, and 37 ~ C incubation recommenced. Individual tubes were removed from the water bath at timed intervals and placed on ice, and 1.5 ml ice-cold 0.02 mol/L EDTA PBS was added. Tubes were centrifuged at 4 ~ C for 5 minutes at 150g, and the erythrocyte pellet was washed three times with cold 0.02 mol/L EDTA PBS. Radioactivity in the pellet was counted in an LKB gamma counter (LKB Diagnostics Inc., Gaithersburg, Md.), and compared with total reactivity input. Nonspecific counts (radioactivity present in samples containing only human erythrocytes and complexes, or human erythrocytes, complexes, and serum in EDTA) were subtracted in all studies. Preliminary experience with this system, adapted from one of Baatrup et al., 2! showed that a maximum of about 370 molecules BSA per human erythrocyte could be bound. This figure compares favorably with the reported numbers of erythrocyte CR1. RESULTS

C3 serum concentration. The 42 subjects studied included 26 with the C3S phenotype, 3 with C3F, and 13 with C3SF. Mean serum C3 concentrations did not differ significantly in these groups (means [___SD]: C3S = 146 _+ 24; C3F = 141 + 33; C3FS = 141 __+22). C3 hemolytic efficiency. In triplicate experiments the mean hemolytic efficiency of C3S was 112% and that of C3F 85% (with PNHS defined as 100%). Correspondingly, 1251 monoclonal anti-C3c binding showed that 1.4 times more C3S than C3F bound to the EAC142 complex (Table I). C3b control by regulators. Trypsinized C3S or C3F (C3b) incubated with factor I and increments of factor H showed no differences in a prime chain cleavage (Fig. 1, A). Sim ilarly, no differences were evident when C3S or C3F preparations were treated with factor I and increments of human erythrocytes as a source of CR1 (Fig. 1, B). C3 support for immune complex solubilization. Solubilization of 1251BSA-anti-BSA complexes was determined in

Volume 116 Number 5

Functional consequences o f polymorphism

S95

100 r

p. z

uJ

n" uJ n CO

IO0

V

8O

o

z

IJ.

0

z_

6O

r/////////J 1/111111114

z rn 0

1/111/11171 ..........

Ii

~ffH/Ha

< "r" 0

40 1.1.. 0 LLI L9 < > < UJ ._1 0

80

!

o UJ D. 60

A

0.008

0.016

60

0.031

).063

,/;'

40

0.125 tO

H ADDED (ug PER ug C 3 b )

i

20

z mLU 0. O9

100

0

I

~ '

80

o

~

........

r

F~f~

.......

~/////A

.........

~/II//4

LI.

0 z < -10

0 m ~ < < _J 0

60

- ~1~4

,~
Fill/IliA

Y///IIA

viii/ill,

VA

40

~

I

I

10

15

TIME (minutes) Fig. 2. BSA-anti-BSA complexes labeled with 1251were solubilized in presence of excess C3S or C3F and human erythrocytes. Bound radioactivity was counted at timed intervals and expressed as percentage of total radioactivity bound, less nonspecificbinding. Each point represents mean of four separate experiments.

~HHH,

20

1.1

B

I

5

2.2

4.4

8.8

17.6

RBC A D D E D ( 107pER ug C3b)

Fig. t. C3S (open bars) or C3F (hatched bars) preparations were incubated with complementfactor I and incrementsof factor H (A) or with human erythrocytes (RBC) (B). Cleavage of a chain of C3 was then assessed by densitometric analysis of SDS-PAGE gels. No differences were evident with either system. Data represent means of determinations. (A, Factor H measured in micrograms per microgram of C3b; B, erythrocytes measured as 107 per microgram of C3b.)

PNHS reconstituted with C3S or C3F. In triplicate experiments, solubilization was equal at 30 and" 120 minutes (Table II). Uptake of solubilized complexes on human erythrocytes.

Figure 2 shows the mean values of four separate studies of immune complex solubilization and uptake onto human erythrocytes. At 15 minutes the uptake of radiolabel onto erythrocytes was nearly identical in systems to which excess C3S or C3F had been added. Furthermore, there was no evidence that the kinetics of solubilization or erythrocyte uptake was enhanced by one or the other allotype. DISCUSSION Inherited polymorphism has been demonstrated for many of the components of the complement system. In the case of C4, this polymorphism is extensive and is associated with differing affinities of C4b for surfaces. 22 Unlike the situation with C4, two allotypes account for 99% of C3 phenotypes; in most populations studied, the C3"S frequency is 0.8. Alper and Propp, ] in the original description of the polymorphism, found no significant differences in hemolytic activity per milligram of protein in 13 subjects of varying C3 phenotype.

S96

Welch, Beisehel, and Kleesattel

The affinity of the two C3 alleles for complement receptors was examined in 1974 by Arvilommi.2 This investigator coated erythrocytes with C3, using sera homozygous for C3S and C3F, and measured the ability of these cells to form rosettes with human mononuclear cells. The C3F erythrocytes showed a somewhat greater affinity for monocytes than their C3S counterparts, but considerable overlap occurred. C3 was affixed to erythrocytes by low ionic strength incubation, and there was no quantitation of binding. Despite the paucity of data pointing to important functional differences between these allotypes, several studies have invoked such differences to account for distortions of C3 allele or phenotype frequency in disease. 3-s In none of these situations, however, is the association particularly strong. In our study, we examined a number of aspects of C3 function that could be relevant to association with disease. We first examined the relationship between C3 phenotype and the serum concentration of the protein, in view of an earlier report suggesting reduced concentration of the C3F protein. 1The serum concentration of C3, although variable, was not a function of C3 phenotype. When a hemolytic system with purified components was used, C3S had a slightly greater (1.4) hemolytic efficiency than C3F. As with the C4 system, 22 this difference paralleled differences in binding efficiency as measured by C3b uptake onto the erythrocyte surface. Given the range of C3 serum concentrations, however, this minor difference should not affect the total hemolytic capacity of serum. After C3b is deposited on a surface, its continued activity is limited by the regulatory proteins H, I, and CR1. We observed no important differences between C3S and C3F in their ability to interact with control proteins. C3b (F) and C3b (S), when incubated with constant amounts of factor I and increasing concentrations of factor H or CR1, demonstrated similar degrees of conversion to C3bi. C3b has a number of functions in addition to its role in forming a C5 convertase and initiating activation of the terminal complement components. The protein can become intercalated within an antigen-antibody complex, breaking the complex into smaller components, an alternative pathway process referred to as immune complex solubilization.2~ C3b coated complexes, in turn, attach via the CR1 to erythrocytes in preparation for further processing.23 Defective solubilization has been reported in a number of diseases.20, 24 Using preformed immune complexes with radiolabeled antigens, we were unable to demonstrate any differences in solubilization of complexes by serum containing either C3S or C3F. Furthermore, the uptake by erythrocytes of immune complexes solubilized by C3S or C3F was equal.

The Journal of Pediatrics May 1990

Recent studies employing R N A / R N A hybridization have localized the difference between the C3F and C3S allotypes to the C3d region of the molecule. Sequencing studies have further shown that a single nucleotide substitution in codon 1216 apparently is responsible for the C3 allotypic difference. This polymorphism results in an asparagine in eodon 1216 in the C3S molecule and an aspartic acid in C3F. 24 This position is 207 amino acids from the thiol ester binding site, toward the carboxy terminal of the c~chain. 25 It is not known whether, in the tertiary structure of the C3 molecule, this amino acid is brought into close enough proximity to this site to affect binding affinity. A less conservative substitution at amino acid 1106 in the C4 molecule has been shown to produce the surface-binding differences of this protein (C4A: aspartic acid; C4B: histidine).26 Although this location is 115 amino acids from the C4 thiol ester site, 27 the tertiary structure of the molecule presumably brings it into a configuration by which it can affect the relative affinity of C4A and C4B for aminoor carboxy-rieh surfaces. Studies examining the binding of hybrid C3 molecules, constructed with differing amino acids at position 1216, could establish the importance of this site in the functional differences we have recognized. It must be stressed, however, that the magnitude of the C3 allotypic binding differences is only about one third of that observed for C4. Thus we found no evidence that the genetic polymorphism of C3 influenced physiologically important functions of the molecule. We conclude that this polymorphism is probably of no important consequence to the organism's immune state. In the absence of a clearly defined hypothesis, we do not believe that further epidemiologic surveys of C3 genetics in specific diseases are warranted. REFERENCES

1. Alper CA, Propp RP. Genetic polymorphismof the third component of human complement (C3). J Clin Invest 1968;47: 2181-91. 2. ArvilommiH. Capacity of complementC3 phenotypesto bind on to mononuclear cells in man. Nature 1974;251:740-1. 3. Wyatt RJ, Julian BA, Galla JH, McLean RH. Increased frequency of C3 fast alleles in IgA nephropathy. Disease Markers 1984;2:419-28. 4. Papiha SS, Rodger RSC. C3 and Bf complement types in chronic renal failure. Hum Genet 1986;72:260-1. 5. Farhud DD, Ananthakrishnan R, Walter H. Association between C3 phenotypes and various diseases. Humangenetik 1972;17:57-60. 6. Dissing J, Lund J, Sorensen H. C3 polymorphism in a group of old arteriosclerotic patients. Hum Hered 1972;22:466-72. 7. Sehiotz PO, Hoiby N, Morling N, Sorensen H. C3 polymorphism in a Danish cystic fibrosis population and its possible association with antibody response. Hum Hered 1978;28:293300. 8. Bronnestam R. Studies of the C3 polymorphism.Relationship

Volume 116 Number 5

9. 10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

between C3 phenotypes and rheumatoid arthritis. Hum Hered 1973;23:206-13. Welch TR, Berry A. C3 alleles in diseases associated with C3 activation. Disease Markers 1987;5:81-7. Dahlqvist SR, Beckman G, Beckman L. Bf and C3 complement types in rheumatoid arthritis. Hum Hered 1985;35: 240-5. Lundwall A, Malmheden I, Stalenheim G, Sjoquist J. Isolation of component C4 of human complementand its polypeptide chains. Eur J Biochem 1981;117:141-6. Hammer CH, Wirtz GH, Renfer L, Gresham HD, Tack BF. Large scale isolation of functionally active components of the human complement system. J Biol Chem 1981;256:3995-4006. Gardner WD, White P J, Hoch SO. Identification of a major human serum DNA-binding protein as beta-lH of the alternative pathway of complement activation. Biochem Biophys Res Commun 1980;94:61-7. March SC, Parikh I, Cuatrecasas P. A simplified method for cyanogen bromide activation of agarose for affinity chromatography. Anal Biochem 1974;60:149-52. Gigli I, Porter RR, Sim RB. The inactivated form of the first component of human complement, C1. Biochem J 1976; 157:541-8. Mayer MM. Complement and complement fixation. In: Kobat EA, Mayer MM, eds. Experimental immunochemistry. Springfield, II1.: Charles C Thoma s, 1961:133ff. Policy M J, Muller-Eberhard HJ. Enhancement of the hemolytic activity of the second component of human complement by oxidation. J Exp Med 1967;120:1013-25. Sim RB, Malhotra V, Day AJ, Erdei A. Structure and specificity of complement receptors. Immunol Lett 1987;14:18390. Laemmli UK. Cleavage of structural proteins during the

Functional consequences, o f polymorphism

20.

21.

22.

23.

24.

25.

26.

27.

S 97

assembly of the head of bacteriophage T4. Nature 1970; 227:680-5. Welch TR, Kleesattel A, Beischel L. Inhibition of immune complex solubilization by sera of patients with membranoproliferative glomerulonephritis. Clin Exp Immunol 1988;72: 103-7. Baatrup G, Petersen I, Kappelgaard E, Jepsen HH, Svehag S-E. Complement-mediated solubilization of immune complexes: solubilization inhibition and complement factor levels in SLE patients. Clin Exp lmmunol 1984;55:313-8. Isenman DE, Young JR. The molecular basis for the difference in immune hemolysis activity of the Chido and Rodgers isotypes of human complement component C4. J Immunol 1984;132:3019-27. Jepsen HH, Svehag S-E, Jarlbaek L, Baatrup G. Interaction of complement-solubilized immune complexes with CR1 receptor on human erythrocytes: the binding reaction. Seand J Immunol 1986;23:65-73. Poznansky MC, Clissold PM, Lachmann PJ. The difference between human C3F and C3S results from a single amino acid change from an asparagine to an aspartate residue at position 1216 on the a-chain of the complement component, C3. J Immunol 1989;143:1254-8. De Bruijn MHL, Fey GH. Human complement component C3: cDNA coding sequence and derived primary structure. Proc Natl Acad Sci USA 1985;82:708-12. Carroll MC, Fathallah DM, Bergamaschino L, Alicot EM, Isenman DG. The chemical basis for the functional hemolytic difference between the two isotypes of human C4 [Abstract]. FASEB J 1989;3:A367. Belt KT, Carroll MC, Porter RR. The structural basis of the multiple forms of human complement component C4. Ceil 1984;36:907-14.