The human C3b/C4b receptor (CR1) is a (5)(6)(7)(8). Initial identification of a partial CR1 cDNA has allowed localization of this complement regulatory gene complex to the q32 region of chromosome 1 (9). More recently, the complement receptor for C3d (CR2) was shown to be structurally related to CR1 (10).An interesting and unusual allelic polymorphism of CR1 based on molecular weight differences has been described (11-14). The four allelic variants have molecular weights of 190,000 (CR1-A), 220,000 (CR1-B), 160,000 (CR1-C), and 250,000 (CR1-D) and are found with a gene frequency of 0.83, 0.16, 0.01, and 0.002, respectively. The structural basis for this polymorphism is incompletely understood but appears to be due to peptide differences rather than posttranslational modifications (15). This conclusion is based on results (15) that demonstrated the following: (i) deglycosylation with endoglycosidase F decreases the molecular weight by -20,000 for all four variants, (ii) biosynthetic labeling of all four allotypic CR1 variants in Epstein-Barr virus (EBV)-transformed cell lines identifies pro-CR1 forms that retain the molecular weight differences of the mature CR1 molecules, and (iii) CR1 contains only N-linked carbohydrates and no sulfate, phosphate, or lipids.Methods to purify CR1 to homogeneity at the 1-to 10-nM level have been described by ourselves (16) and others (17,18). The NH2 terminus of CR1 is blocked (16,17). In order to obtain sequence of internal tryptic peptides, a method was developed to rapidly isolate and sequence individual peptides. A 2.5-kilobase (kb) cDNA clone for CR1 was identified and characterized. The molecular nature of the CR1 allelic molecular weight polymorphism was assessed by an analysis of mRNA of cells expressing the CR1 phenotypes.
MATERIALS AND METHODSPurification of CR1 and Tryptic Peptide Fragments. Four nanomoles of erythrocyte CR1 (16) was reduced in 0.1 M NH4HCO3/1% NaDodSO4/20 mM dithiothreitol at 370C for 90 min and then alkylated in the dark by the addition of50 mM iodoacetic acid at 370C for 60 min. After precipitation overnight at -20'C with a 10-fold excess (vol/vol) of methanol and a 2% (wt/wt) amount of L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (TPCKtrypsin), the preparation was centrifuged at 25,000 x g at 40C for 30 min. The supernatant was removed, the tube was dried under N2, and CR1 was resolubilized in 0.5 ml of 0.1 M NH4HC03/0.001% NaDodSO4/2 mM CaCl2. TPCK-trypsin (2%, wt/wt) was added and then again at 4 hr. After incubation at 370C for a total of 16 hr, the sample was lyophilized.A "two-dimensional" HPLC separation of tryptic fragments was performed, using a Waters liquid chromatograph.Abbreviation: EBV, Epstein-Barr virus.
2459The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.