The quantitative expression of complement receptor type 1 (CR1) on erythrocytes is regulated by two CR1 alleles that differ in having genomic HindIII fragments of either 7.4 or 6.9 kb and that determine high (H allele) or low (L allele) CR1 expression, respectively, across a 10-fold range. To investigate whether the product of the L allele may contain amino acid substitutions that make it more susceptible to proteolysis, cDNA sequence spanning the CR1 coding region was analyzed in two donors who were homozygous for the H and L alleles and differed by 7-fold in their mean numbers of CR1 per erythrocyte. Sequence differences were detected at 10 nucleotide positions, including 6 that would cause amino acid substitutions. The HindIII RFLP and 3 of the latter 6 sites were analyzed in genomic DNA of 85 Caucasians and 75 African Americans; sites encoding the other amino acid substitutions were analyzed less extensively. Two major haplotypes defined prototypic H and L alleles in both ethnic groups, suggesting that these alleles existed before the African and European populations diverged. Decreased erythrocyte CR1 expression is associated with impaired clearance of immune complexes from blood. Persistence of the L allele in all populations that have been analyzed may suggest a compensatory survival advantage, perhaps related to malaria or another infectious disease.

Removal of complement-activating particles and immune complexes from blood depends on the function of complement receptor type 1 (CR13; CD35) of erythrocytes (1, 2, 3). CR1 is a large, single-chain molecule (Mr ∼200,000) that can bind C3b, C4b, and C1q (1, 2, 3, 4). The latter molecules function in innate and adaptive immunity by attaching to the targets of immune reactions, focusing further complement activation at these sites and opsonizing the targets for binding by erythrocyte CR1 (4, 5, 6). Complexes that have bound to CR1 are transferred to phagocytes as erythrocytes traverse the liver and spleen (7, 8, 9, 10).

Quantitative expression of CR1 on erythrocytes is regulated by a genetic element that is linked to the site of a HindIII RFLP of the CR1 gene (11). Two alleles that are associated with either high (H allele) or low (L allele) expression of CR1 differ in having genomic HindIII fragments of 7.4 and 6.9 kb, respectively. Individuals homozygous for the H allele can have more than 1000 CR1 molecules per erythrocyte, whereas those homozygous for the L allele generally have fewer than 200 CR1 per cell; heterozygous individuals have intermediate CR1 expression. Correlation between the HindIII RFLP and the quantity of CR1 on erythrocytes has been confirmed in multiple populations on several continents (12, 13, 14, 15). A significant correlation was not observed, however, among African Americans, despite a trend in the expected direction (15).

As erythrocytes age in the circulation CR1 is lost through proteolysis (16, 17, 18), perhaps during transfer of bound immune complexes to reticuloendothelial phagocytes (19). In initial studies of age-fractionated erythrocytes, groups of donors homozygous for either the H or L allele did not appear to differ in their rates of loss of erythrocyte CR1 (20). It was subsequently found that donor-dependent differences in CR1 expression were already evident in reticulocytes and that erythrocyte CR1 loss was actually faster in donors with higher CR1 expression (21). However, erythroid cells develop in intimate contact with macrophages that may modify their surface proteins (22, 23), and circulating erythrocytes with low CR1 numbers are less likely to bind immune complexes (24, 25, 26) and transfer them to phagocytes. Thus, rapid proteolysis of CR1 during erythroid development could result in its more gradual loss from circulating erythrocytes. When age-fractionated erythrocytes from donors heterozygous both for the H and L alleles and for different CR1 structural allotypes were analyzed in Western blots, it was found that the product of the L allele may in fact be lost more rapidly during erythrocyte aging (15). The H and L allelic products of heterozygous donors would presumably have similar exposure to immune complex binding and transfer reactions. Further, analyzing these allelic products simultaneously in Western blots has the advantage of controlling internally for variations in erythrocyte fractionation and protein analysis. The fact that the H and L allelic products were lost from erythrocytes of one donor at apparently equal rates (15) may indicate that some individuals lack a relevant protease or that linkage disequilibrium between the HindIII RFLP and the element that regulates CR1 expression is incomplete.

The H and L alleles are identical in the sequences of their transmembrane and cytoplasmic domains (15). Thus, any amino acid substitutions causing allelic differences in susceptibility to proteolysis would have to be extracellular. The extracellular domain of CR1 is comprised of a series of long homologous repeats (LHRs), each containing seven short consensus repeats (SCRs) (Fig. 1) (27, 28, 29). The most common structural allele of CR1 has four LHRs, but alleles known or presumed to have three, five, or six LHRs also exist (1, 2). Very high homology among the LHRs complicates efforts to amplify selected coding regions specifically, and highly homologous repeats also span extended regions of CR1 genomic DNA (30, 31, 32). In this study, an approach was developed that allows amplification and direct sequencing of overlapping fragments entirely spanning the CR1 coding sequence. The complete coding sequence of representative H and L alleles was determined, polymorphisms distinguishing these alleles were identified, and haplotypes defined by these polymorphisms were analyzed in groups of Caucasian and African American donors.

FIGURE 1.

Analysis of the CR1 coding sequence by RT-PCR. The 5′- and 3′-untranslated (UT) regions, LHR-A, -B, -C, and -D, and the predicted transmembrane (TM) and cytoplasmic (C) domains of CR1 cDNA are shown at the top of the figure. The numbered squares beginning beneath LHR-A represent the first 10 of 30 SCRs that comprise the extracellular domain of CR1, including 7 SCRs in each LHR and 2 additional SCRs 3′ to LHR-D. The 4 numbered solid lines depict RT-PCR products that were amplified directly from cDNA. The solid triangles and bars beneath product 2 indicate the locations of BsrDI and PstI sites, respectively. Gel-purified restriction fragments of product 2 were used as templates for the production of the 5 smaller PCR products that are depicted by the numbered dotted lines. The nucleotide positions of the nine PCR products in CR1 cDNA were: product 1, −74 to 490; product 2, 304-4213; product 3, 4023–4916; product 4, 4554–6328; product 5, 345-1050; product 6, 1088–2318; product 7, 2955–4213; product 8, 863-1449; and product 9, 2208–3193. Nucleotides are numbered as described (32 ).

FIGURE 1.

Analysis of the CR1 coding sequence by RT-PCR. The 5′- and 3′-untranslated (UT) regions, LHR-A, -B, -C, and -D, and the predicted transmembrane (TM) and cytoplasmic (C) domains of CR1 cDNA are shown at the top of the figure. The numbered squares beginning beneath LHR-A represent the first 10 of 30 SCRs that comprise the extracellular domain of CR1, including 7 SCRs in each LHR and 2 additional SCRs 3′ to LHR-D. The 4 numbered solid lines depict RT-PCR products that were amplified directly from cDNA. The solid triangles and bars beneath product 2 indicate the locations of BsrDI and PstI sites, respectively. Gel-purified restriction fragments of product 2 were used as templates for the production of the 5 smaller PCR products that are depicted by the numbered dotted lines. The nucleotide positions of the nine PCR products in CR1 cDNA were: product 1, −74 to 490; product 2, 304-4213; product 3, 4023–4916; product 4, 4554–6328; product 5, 345-1050; product 6, 1088–2318; product 7, 2955–4213; product 8, 863-1449; and product 9, 2208–3193. Nucleotides are numbered as described (32 ).

Close modal

Southern blots of HindIII-digested genomic DNA were probed with the pUC18 subclone pHL1.4 (kindly provided by Dr. Winnie Wong), having an insert that hybridizes specifically to the genomic fragments that define the HindIII RFLP of CR1.

RNA was isolated with a Rapid Total RNA Isolation Kit (5[prime → 3′, Boulder, CO) from PBMC of two Caucasian donors, one homozygous for the H allele of CR1 and having a mean of 1050 CR1 per erythrocyte and the other homozygous for the L allele and having 140 CR1 per erythrocyte. Cellular CR1 was quantitated by the binding of 125I-YZ-1 anti-CR1 (33, 34). Overlapping fragments spanning the coding sequence of CR1 were amplified by RT-PCR (Fig. 1, Table I). Four primary products (Fig. 1, fragments 1–4) were produced directly from cDNA under the conditions shown (Table I). Amplification of fragments 1 and 3 was performed with a GeneAmp XL RNA PCR Kit (Perkin-Elmer, Foster City, CA); cDNA synthesis was primed by the reverse amplimer shown for each fragment in Table I. cDNA for the production of fragment 2 was synthesized with the SuperScript Preamplification System (Life Technologies, Gaithersburg, MD) primed by the reverse amplimer for this fragment. Amplification of fragment 2 was performed with a GeneAmp XL PCR Kit and a “hot start” technique, as described by the manufacturer for the control template DNA. The conditions described for fragment 2 (Table I) did not yield a single, homogenous product but produced a major fragment of 3910 bp that was isolated from minor contaminating fragments by excision and electroelution from agarose gels. Fragment 4 was prepared as described (15).

Table I.

Amplification of CR1 cDNA and genomic sequencesa

FragmentAmplimersSize (bp)TA (°C)
RT-PCR 5′-CGCGGAGCACAATGATTGGTCACTCCTAT-3′ 564 63b 
↓  5′-CCCAAATGACAGTATCACCTGAGATGATGC-3′   
 5′-ACTGGTGCTAAGGACAGGTGCAGACGT-3′ 3910 69c 
  5′-AGTGACCAGCACGAACAGAAAGTTC-3′   
 5′-CACAGGAACTCCCTCTGGAGATATTCCCTA-3′ 894 63b 
  5′-GTGTGGGACCCTACCATGACAAACCCG-3′   
 5′-GAAGGCACCTATTTGTGAGATCATA-3′ 1775 61d 
  5′-AAAGAGGCAGGAGCTTTGCTAGG-3′   
Reamplification 5′-TCCTCCAGATCCTGTGAATGGC-3′ 706 58 
of restriction fragments  5′-TGTGCAGCGCATAGACGCA-3′   
↓ 5′-AAGTGAAATCCTGTGATGACTTCATGGGCC-3′ 1231 60e 
  5′-TCCTTGTCCCTTTGGGTACGCTCAGC-3′   
 5′-GTACTACGGGAGGCCATTCTCTATCA-3′ 1259 61e 
  5′-AGTGACCAGCACGAACAGAAAGTTCA-3′   
 5′-CAGGCCCTGAACAAATGGGAGC-3′ 587 63e 
  5′-AGGGTCACTTGTGCAGCGGATG-3′   
 5′-TGTGAAGTGCCAGGCCCTGAA-3′ 986 63e 
  5′-TGCTCCAATGGGCAGTATTGCC-3′   
Genomic PCR Exon 9 5′-TGCAGATACTCTGGTGAACTCC-3′ 308 60 
↓  5′-TGAACCCATTAGGGATGTGG-3′   
 Exon 13 5′-TCACTCAGCCTGGTACAGAGCA-3′ 529 62 
  5′-TGATCCAACAGCAACGCATG-3′   
 Exon 19 5′-TGCGTTGGATCTTTCCCATG-3′ 892 63 
  5′-CCTGGTTTCCAGCAAGGATACA-3′   
 Exon 22 5′-TTCACATTGGATAGCCAGAGC-3′ 682 63 
  5′-CCAGAGGTTAATCTCCCTGGA-3′   
 Exon 29 5′-GCAGAACCCATATCATACACACCT-3′ 633 60 
  5′-CTCACACCCAGCAAAGTCTTGA-3′   
 Exon 33 5′-AAGCGCACAGTCACAGGTCAC-3′ 443 59 
  5′-GGAGGTAGTTCTGTCTCTGAC-3′   
 HindIII RFLP 5′-CAGCAGAGCCAACTTCTGACCC-3′ ∼1.7 kb 54e 
 (intron 27) 5′-CCCTTGTAAGGCAAGTCTGG-3′   
FragmentAmplimersSize (bp)TA (°C)
RT-PCR 5′-CGCGGAGCACAATGATTGGTCACTCCTAT-3′ 564 63b 
↓  5′-CCCAAATGACAGTATCACCTGAGATGATGC-3′   
 5′-ACTGGTGCTAAGGACAGGTGCAGACGT-3′ 3910 69c 
  5′-AGTGACCAGCACGAACAGAAAGTTC-3′   
 5′-CACAGGAACTCCCTCTGGAGATATTCCCTA-3′ 894 63b 
  5′-GTGTGGGACCCTACCATGACAAACCCG-3′   
 5′-GAAGGCACCTATTTGTGAGATCATA-3′ 1775 61d 
  5′-AAAGAGGCAGGAGCTTTGCTAGG-3′   
Reamplification 5′-TCCTCCAGATCCTGTGAATGGC-3′ 706 58 
of restriction fragments  5′-TGTGCAGCGCATAGACGCA-3′   
↓ 5′-AAGTGAAATCCTGTGATGACTTCATGGGCC-3′ 1231 60e 
  5′-TCCTTGTCCCTTTGGGTACGCTCAGC-3′   
 5′-GTACTACGGGAGGCCATTCTCTATCA-3′ 1259 61e 
  5′-AGTGACCAGCACGAACAGAAAGTTCA-3′   
 5′-CAGGCCCTGAACAAATGGGAGC-3′ 587 63e 
  5′-AGGGTCACTTGTGCAGCGGATG-3′   
 5′-TGTGAAGTGCCAGGCCCTGAA-3′ 986 63e 
  5′-TGCTCCAATGGGCAGTATTGCC-3′   
Genomic PCR Exon 9 5′-TGCAGATACTCTGGTGAACTCC-3′ 308 60 
↓  5′-TGAACCCATTAGGGATGTGG-3′   
 Exon 13 5′-TCACTCAGCCTGGTACAGAGCA-3′ 529 62 
  5′-TGATCCAACAGCAACGCATG-3′   
 Exon 19 5′-TGCGTTGGATCTTTCCCATG-3′ 892 63 
  5′-CCTGGTTTCCAGCAAGGATACA-3′   
 Exon 22 5′-TTCACATTGGATAGCCAGAGC-3′ 682 63 
  5′-CCAGAGGTTAATCTCCCTGGA-3′   
 Exon 29 5′-GCAGAACCCATATCATACACACCT-3′ 633 60 
  5′-CTCACACCCAGCAAAGTCTTGA-3′   
 Exon 33 5′-AAGCGCACAGTCACAGGTCAC-3′ 443 59 
  5′-GGAGGTAGTTCTGTCTCTGAC-3′   
 HindIII RFLP 5′-CAGCAGAGCCAACTTCTGACCC-3′ ∼1.7 kb 54e 
 (intron 27) 5′-CCCTTGTAAGGCAAGTCTGG-3′   
a

Amplification was performed in a GeneAmp PCR System 9600. Except where noted, conditions were 94°C for 60 s, then 35–40 cycles of 94°C for 15 s, TA (annealing) for 15 s, and 72°C for 60 s (extension). Mg2+ concentrations (mM) were 0.86 (fragments 1 and 3), 1 (fragment 2), 1.75 (fragment 4), 3 (fragments 5–9 and exon 9), 2 (exon 13), and 1.5 (exons 19, 22, 29, and 33, and HindIII RFLP/intron 27).

b

Forty cycles of 94°C for 15 s and 63°C for 60 s (annealing/extension).

c

Twenty cycles of 94°C for 15 s and 69°C for 180 s; then 20 cycles in which the 69°C incubation was extended by 15 s, incrementally, for each successive cycle.

d

As described (15 ).

e

Extension phase: 90 s (fragments 6, 7); 75 s (fragments 8, 9); or 120 s (HindIII RFLP/intron 27).

Highly homologous repeats occurring throughout fragment 2 precluded the identification of internal sequencing primers or amplimers that hybridized at unique sites within this fragment. Thus, templates for secondary amplification reactions (Fig. 1, fragments 5–9) were produced by digesting the gel-purified fragment 2 of each donor with either PstI (fragments 5–7) or BsrDI (fragments 8 and 9) (New England BioLabs, Beverly, MA). Desired restriction fragments were gel purified, and selected regions were amplified under the conditions shown (Table I).

Products amplified from cDNA were analyzed by automated sequencing, with internal primers derived from the published sequence of CR1 (GenBank accession number Y00816) (29). Primers were spaced so that each cDNA region was covered by multiple sequencing reactions. Genomic sequences were analyzed with a Sequenase 7-Deaza-dGTP DNA Sequencing Kit (United States Biochemical, Cleveland, OH) or a SequiTherm EXCEL II DNA Sequencing Kit (Epicentre Technologies, Madison, WI).

Exons containing polymorphic sites detected in CR1 cDNA (Table II) were amplified from genomic DNA of Caucasian and African American donors under the conditions shown in Table I. Amplimers were derived from published sequences of introns flanking these exons (GenBank accession numbers L17390–L17430) (32). Polymorphisms at CR1 nucleotide positions 1360, 2078, and 4870 were analyzed by direct sequencing. The polymorphisms at positions 3093 and 3650 alter restriction sites for BstNI (New England BioLabs) and RsaI (Boehringer Mannheim, Indianapolis, IN), respectively; genotypes at these sites were determined by restriction analysis of PCR fragments spanning the sites. BstNI fragments of the exon 19 PCR product shown in Table I have the following sizes (base pairs): G3093: 566, 230, 91; T3093: 566, 321. Multiple sets of primers and conditions for amplifying exon 19 also produced a comigrating fragment that resembled the G3093 allotypic fragment with respect to its cleavage by BstNI. This finding undoubtedly reflects the occurrence of highly homologous genomic repeats within CR1 or a CR1-related sequence (28, 31, 35). Heterozygosity at nucleotide 3093 was thus distinguished from homozygosity for T3093 by differences in band density in ethidium bromide-stained gels. Independent interpretations of the stained gels by two individuals were completely concordant. RsaI fragments of the exon 22 PCR product (Table I) have the following sizes (base pairs): A3650: 520, 162; G3650: 458, 162, 62. The nucleotide substitution at position 5507 alters a restriction site for MnlI (New England BioLabs). This site was analyzed either by direct sequencing of the exon 33 PCR product described in Table I or by restriction analysis of a 305-bp fragment produced with the same forward primer but with the reverse primer 5′-GAACAGAAAGTTCACAGCGAGG-3′.MnlI fragments of the latter product have the following sizes (base pairs): C5507: 111, 80, 37, 33, 33, 11; G5507: 111, 80, 70, 33, 11; these fragments were resolved in nondenaturing 16% polyacrylamide gels. The HindIII RFLP in CR1 intron 27 was analyzed by Southern blotting or by a modification of described methods (36). HindIII fragments of the intron 27 PCR product shown in Table I have the following sizes (base pairs): H allele, ∼1600, 84; L allele, ∼1,150, 465, 84.

Table II.

Polymorphisms detected in the coding sequence of CR1

NucleotideaExonAmino AcidSCR
A207Gb, c Silent in Glu60 
T981Cd Silent in Pro318 
A1356G Silent in Gly443 
A1360G T445A 
T2078Cc 13 I684T 10 /11 
T2367Cc 14 Silent in Tyr780 12 
G3093Tc 19 Q1022H 16a 
A3650G 22 H1208R 19 
A4870Gc, e 29 I1615V 25 
C5507Gc, e 33 P1827R 28 
C5654Tc, e 34 T1876I 29 
NucleotideaExonAmino AcidSCR
A207Gb, c Silent in Glu60 
T981Cd Silent in Pro318 
A1356G Silent in Gly443 
A1360G T445A 
T2078Cc 13 I684T 10 /11 
T2367Cc 14 Silent in Tyr780 12 
G3093Tc 19 Q1022H 16a 
A3650G 22 H1208R 19 
A4870Gc, e 29 I1615V 25 
C5507Gc, e 33 P1827R 28 
C5654Tc, e 34 T1876I 29 
a

Nucleotides are numbered as described (32 ).

b

Nucleotides identical with those at the corresponding position in 220-kDa chimpanzee CR1 (43 ) are underlined and bold.

c

Reported previously (32 ).

d

Reported previously (35 ).

e

Reported previously (15 ).

Donors were classified by the investigators as African American or Caucasian based on appearance and were not questioned regarding their ancestry. Prior analysis suggests up to 25% European genetic admixture among African Americans (37). Genomic DNA was obtained from Caucasian donors in the Jackson, MS, and Boston, MA, areas. All African American donors were from Jackson, MS.

The coding sequence of the H and L alleles of CR1 was determined by analyzing cDNA of two Caucasian donors who were homozygous for opposite alleles. Comparison of the observed sequences with each other and with the published coding sequence of CR1 (27, 28, 29, 32, 35) revealed 11 polymorphic sites (Table II). Several of these polymorphisms had been detected previously (15, 32, 35), and the C5507G substitution has been shown to be specific to the L allele (15). Both donors in the current study were homozygous for “T” at position 5654, which was previously reported as “C” (27); thus, this polymorphism is not specific with respect to the H and L alleles. The nucleotide substitutions at positions 207, 981, 1356, and 2367 would not result in amino acid substitutions, and their possible allele specificity was not assessed.

The nucleotide substitutions at positions 3093, 3650, and 5507 in CR1 cDNA alter restriction sites for BstNI, RsaI, and MnlI, respectively. These sites and that of the HindIII RFLP were analyzed in genomic DNA of 85 Caucasian and 75 African American donors (Table III). Findings regarding the HindIII RFLP and C5507G polymorphisms of some of these donors have been reported previously (11, 15). In the samples analyzed, the frequencies of the L allele as identified by the HindIII RFLP were 0.2 and 0.23 for Caucasians and African Americans, respectively, similar to previous findings (11, 15). However, because these samples included a mixture of Caucasian donors from the Jackson and Boston areas and because data for the HindIII RFLP were already known for some donors, these should not be considered random samples. The G3093T and A3650G substitutions, like the C5507G substitution (15), were largely specific to the L allele identified by the HindIII RFLP. Thus, two predominant haplotypes were observed in both Caucasians and African Americans: an H haplotype containing G3093, A3650, and C5507 (encoding Gln1022, His1208, and Pro1827); and an L haplotype containing T3093, G3650, and G5507 (encoding His1022, Arg1208, and Arg1827).

Table III.

CR1 genotypes and deduced haplotypes of Caucasian and African American donors

Nucleotide 3093 (exon 19)Nucleotide 3650 (exon 22)HindIII (intron 27) (kb)Nucleotide 5507 (exon 33)No. Observed (Caucasian/African American)
Genotypesa GG  AA  7.4–7.4  CCb 55 /41 
 GG  AA  7.4–7.4  CGc 2 /0 
 GT  AG  7.4–7.4  CC 1 /1 
 GT  AA  7.4–7.4  CC 0 /2 
 GT  AG  7.4–6.9  CGb 14 /16 
 T A 7.4–6.9  C1 /0 
 GT  AG  7.4–6.9  CC 0 /1 
 GG  AG  7.4–6.9  CG 5 /1 
 GG  AG  7.4–6.9  CC 0 /9 
 TT  GG  6.9–6.9  GGb 6 /4 
 TG  GG  6.9–6.9  GG 1 /0 
         
Haplotypes         
→ G  A  7.4  Cd 132 /112 
   7.4 e 2 /0 
  ▾ 7.4  1 /1 
 ▾  7.4  1 /2 
→ T  G  6.9  Gd 28 /24 
   6.9 ▾ 0 /1 
 ▾  6.9  6 /1 
 ▾  6.9 ▾ 0 /9 
Nucleotide 3093 (exon 19)Nucleotide 3650 (exon 22)HindIII (intron 27) (kb)Nucleotide 5507 (exon 33)No. Observed (Caucasian/African American)
Genotypesa GG  AA  7.4–7.4  CCb 55 /41 
 GG  AA  7.4–7.4  CGc 2 /0 
 GT  AG  7.4–7.4  CC 1 /1 
 GT  AA  7.4–7.4  CC 0 /2 
 GT  AG  7.4–6.9  CGb 14 /16 
 T A 7.4–6.9  C1 /0 
 GT  AG  7.4–6.9  CC 0 /1 
 GG  AG  7.4–6.9  CG 5 /1 
 GG  AG  7.4–6.9  CC 0 /9 
 TT  GG  6.9–6.9  GGb 6 /4 
 TG  GG  6.9–6.9  GG 1 /0 
         
Haplotypes         
→ G  A  7.4  Cd 132 /112 
   7.4 e 2 /0 
  ▾ 7.4  1 /1 
 ▾  7.4  1 /2 
→ T  G  6.9  Gd 28 /24 
   6.9 ▾ 0 /1 
 ▾  6.9  6 /1 
 ▾  6.9 ▾ 0 /9 
a

Fragments spanning the designated polymorphic sites were amplified from genomic DNA of each donor, and the PCR products were characterized as described in Materials and Methods. Observed nucleotide(s) or restriction fragments are shown. The number of donors of each race having each combination of markers is presented in the rightmost column. The genotypes observed are grouped according to the form of the HindIII RFLP detected.

b

Three predominant genotypes were detected in both groups of donors, corresponding to homozygosity for either the H or L allele, or heterozygosity.

c

For donors whose genotypes were unlike the three predominant genotypes, putative novel haplotypes (underlined) were identified by assuming that each such donor had one prototypic H or L allele. Within these haplotypes, nucleotides that differ from those of the prototypic alleles are bold italic.

d

Haplotypes of the prototypic H and L alleles are bold and are identified by right arrows.

e

Inverted triangles indicate regions in which crossover events may have occurred between prototypic H and L alleles to produce the less commonly observed alleles.

By assuming that each of the donors represented in Table III has one allele bearing either the prototypic H or L haplotype, the likely features of six less common haplotypes can be deduced (Table III). At least one minor haplotype was observed mainly among Caucasians, whereas another was seen only in African Americans. Each of the minor haplotypes contains regions of continuity that resemble portions of the major H and L haplotypes, suggesting that the minor haplotypes were produced by recombination between prototypic H and L alleles.

Sequences occurring at the other three polymorphic sites shown in Table II, A1360G, T2078C, and A4870G, were analyzed in products amplified from genomic DNA of donors homozygous for the H or L allele, as indicated by the HindIII RFLP. In this more limited analysis, each of the three polymorphisms appeared to be in linkage disequilibrium with the HindIII allelic markers. “A” at position 1360 was observed only in association with the H allele, being present on 6 of 18 H alleles of Caucasians and 2 of 10 H alleles of African Americans; the remaining H alleles and all of 18 L alleles analyzed (10 Caucasian, 8 African American) had a “G” at this site. Among Caucasians, there was complete concordance of T2078 and A4870 with the H allele, and of C2078 and G4870 with the L allele (Table IV). In African Americans, similar allele specificity was observed at position 2078 in 17 of 18 cases. In contrast, “G” was present at position 4870 not only on all of 8 L alleles of African Americans but also on 8 of 10 H alleles.

Table IV.

Allele specificity of the T2078C and A4870G polymorphismsa

2078 (exon 13)HindIII (intron 27) (kb)4870 (exon 29)No. Observed (Caucasian/African American)
7.4 10 /2 
7.4 0 /8 
    
6.9 10 /7 
6.9 0 /1 
2078 (exon 13)HindIII (intron 27) (kb)4870 (exon 29)No. Observed (Caucasian/African American)
7.4 10 /2 
7.4 0 /8 
    
6.9 10 /7 
6.9 0 /1 
a

Fragments spanning the designated polymorphic sites were amplified from genomic DNA of each donor, and the PCR products were characterized as described in Materials and Methods. Observed nucleotides or restriction fragments are shown. The number of CR1 alleles from donors of each race having each combination of markers is presented in the rightmost column. All of the selected donors were homozygous for one of the two forms of the HindIII RFLP.

In this study, the complete CR1 coding sequence was determined in two donors who differed by more than 7-fold in the quantity of CR1 on their erythrocytes; one donor was homozygous for the H allele and had 1050 CR1 per erythrocyte, whereas the other was homozygous for the L allele and had 140 CR1 per cell. Each donor thus had the phenotype that is characteristic of his genotype. Accordingly, if differential expression of the H and L alleles is caused by accelerated proteolysis of the L allotype, cDNA of these two individuals would be predicted to differ at the relevant sites. The sequence of PCR products was determined directly, without a cloning step, thus minimizing the potential effects of misincorporation of nucleotides during amplification. The six polymorphisms that were analyzed further were found in products of genomic DNA of multiple individuals, defining two predominant haplotypes that characterize prototypic H and L alleles of both Caucasians and African Americans (Tables III and IV).

Six of the nucleotide differences between the H and L alleles correspond to predicted amino acid substitutions (Tables II–IV). At least two of these may be biologically insignificant. The substitution of Thr445 to alanine would be associated with slightly increased hydrophobicity within a predicted β sheet region of SCR 7 (38). This SCR is not required for ligand binding (39, 40) and may serve principally as a “spacer” in the extended CR1 molecule. Thus, although it is possible that this substitution could cause exposure of a novel proteolytic cleavage site, it is perhaps more likely to be functionally neutral. I1615V is a conservative substitution at a conserved hydrophobic residue (38); the corresponding amino acid in multiple other SCRs of CR1 is either isoleucine or valine.

Two substitutions, I684T and Q1022H, lie in SCRs that participate in ligand binding (29, 39, 40, 41, 42). Replacement of the tandem isoleucines 683 and 684 by two glutamic acids was reported to reduce ligand binding by a soluble LHR-B construct (40). Whether a single change of Ile684 to threonine, an uncharged polar amino acid, might also affect receptor function is unknown. The position corresponding to Q1022H is occupied by glutamine in all four LHRs of both human and chimpanzee CR1, except in the human L allele (27, 28, 29, 43). An aspartic acid 2 residues upstream of this glutamine (or a corresponding aspartic acid) is important in C3b binding by both human CR1 and the chimpanzee erythrocyte complement receptor (44). Thus, local effects of changing Gln1022 to histidine, which is weakly basic and less hydrophilic than glutamine, could perhaps alter ligand binding by CR1. However, any functional consequences of either the I684T or Q1022H substitutions may be mitigated by the presence of two C3b-binding sites within each CR1 molecule (29) and by the clustered distribution of CR1 on erythrocytes (24, 25, 26, 45).

Each of the other two allele-specific amino acid substitutions, H1208R and P1827R, could have important effects on the structure and stability of CR1. Each would introduce a potential cleavage site for tryptic proteases. H1208R occurs in a region that, by analogy to SCRs 5 and 16 of factor H (46, 47), may be highly solvent exposed, and substitution to arginine would cause increased local hydrophilicity. Similarly, analysis of the SCRs of multiple proteins suggests that P1827R may lie within a surface-exposed turn (38, 48). A change to arginine, having a strongly basic side chain, would not only make this region more hydrophilic but might also be accompanied by a loss of spatial constraints otherwise imposed by proline. Arg1208 and Arg1827 are both present in the prototypic L allele of African Americans, in whom the HindIII RFLP does not correlate tightly with the quantity of CR1 on erythrocytes (15); thus, if either site is the target of a tryptic protease(s) that affects CR1 expression, then deficiency of this protease must be common in African Americans.

These surface-exposed arginine substitutions could perhaps produce novel antigenic epitopes, and thus could potentially contribute to CR1-specific blood group Ags such as the Knops, McCoy, Swain-Langley, and York Ags (49, 50). Antisera defining these blood groups recognize epitopes in both the A (four LHR) and B (five LHR) structural allotypes of CR1, and persons homozygous for each of these allotypes have been identified among producers of the antisera (49, 50, 51). The L allele of CR1 appears to be associated exclusively with the A structural allotype (20, 30). Thus, a person homozygous for the B allotype would also be homozygous for the H allele and would be unlikely to make antiserum against the H allelic product. The fact that these blood group antisera do recognize epitopes in the B (and hence the H) allotype makes it unlikely that they are specific for H/L-related amino acid substitutions.

The prototypic H and L haplotypes of CR1 (Table III) had apparently become established before the divergence of the African and European populations, which probably occurred 100,000–200,000 years ago (52, 53, 54, 55). The minor haplotypes shown in Tables III and IV show further evolution of these alleles in both populations, involving recombination events and perhaps point mutations. All of the minor haplotypes in Table III and the single L allele bearing T2078 in Table IV could be the result of crossover events. On the other hand, A1360 (text, above) and A4870 (Table IV), which were observed largely among Caucasians, probably resulted from point mutations of the H allele. Assuming up to 25% European genetic admixture among African Americans (37), the latter mutations may represent relatively recent changes that occurred in European founders. Comparison of polymorphisms in the H and L alleles to the corresponding sites in the sequence of 220-kDa chimpanzee CR1 (underlined nucleotides in Table II) shows identity between the H allele and chimpanzee CR1 at the two amino acid substitutions in ligand binding regions (I684T and Q1022H) and the two substitutions to arginine (H1208R and P1827R). Thus, evolution of the L allele from an ancestral CR1 sequence probably involved point mutations at each of these sites.

Low expression of erythrocyte CR1 is associated with impaired clearance of immune complexes and with deposition of the complexes outside the reticuloendothelial system (9, 10, 56, 57). Although patients with diseases like systemic lupus erythematosus do not appear to have altered frequencies of the H and L alleles (12, 13, 14, 58, 59, 60), low CR1 expression determined by the L allele could result in increased tissue damage in a large number of inflammatory and infectious conditions. On the other hand, low CR1 expression may provide a selective advantage in other settings. In falciparum malaria, a parasite-encoded protein of infected erythrocytes, PfEMP1, has recently been shown to bind to CR1 of adjacent erythrocytes, producing an agglutination reaction that may cause increased vascular endothelial damage and more severe disease (61). Erythrocytes expressing low numbers of CR1 or having the Sl(a) CR1 polymorphism, which is common in Africans, were found to have reduced binding to PfEMP1 in vitro (61). These considerations make it unlikely that the survival effects of the H and L alleles are entirely neutral. Thus observed frequencies of the L allele in a fairly limited range, from 0.19 to 0.27, in populations as diverse as those of New Delhi and Oulu, Finland (11, 12, 13, 14, 15, 62), may suggest that selective pressures in addition to falciparum malaria have contributed to a balanced polymorphism of the H and L alleles.

Our study has established the coding sequence of the H and L alleles of CR1, identified allele-specific amino acid substitutions that may be functionally significant, and provided information regarding the evolution of these alleles. The methods described may be useful in analyzing additional CR1 polymorphisms, such as those related to Sl(a) and other blood group Ags. If the H and L alleles represent a balanced polymorphism, then identifying the selective pressures responsible for this balance may yield insights into important human diseases.

1

Supported in part by research funds of the Department of Veterans Affairs.

3

Abbreviations used in this paper: CR1, complement receptor type 1; H allele, high allele; L allele, low allele; LHRs, long homologous repeats; SCRs, short consensus repeats.

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