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We examined the interaction of complement component C3 with surface molecules on Trypanosoma cruzi. Five- to six-fold more C3 was bound to epimastigotes (Epi) than to metacyclic trypomastigotes (CMT) of strain M88. Epi and CMT were surface iodinated, then incubated in C8-deficient serum, and detergent lysates were applied to anti-C3 antibody that had been coupled to Sepharose. We found that 9.20-10.24% of applied 125I-Epi protein bound to anti-C3-sepharose, compared to 2.64% binding of 125I-CMT protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that C3 was attached to 125I-Epi protein by a covalent bond. Samples eluted from anti-C3-sepharose with hydroxylamine revealed a single, major, 72 kD band, suggesting that C3b attaches almost exclusively to the 72 kD glycoprotein of Epi by a hydroxylamine-susceptible ester bond. An antiserum was prepared from lysates of serum-treated Epi that had been affinity-purified on anti-C3-sepharose. This antiserum immunoprecipitated a single 72 kD component (gp72) from surface-iodinated Epi, and specifically recognized only gp72 from Epi in immunoblots. In contrast to the results with Epi, gp72 on CMT was not found to be an efficient acceptor molecule for C3 deposition. The results are the first to evaluate the acceptor site for C3 deposition on a parasite, and they show that gp72 on Epi, but not gp72 on CMT, serves as the preferential acceptor for C3 during antibody-independent alternative complement pathway activation.

This is the first report demonstrating that C3d receptor (CR2) has functional activity in regulating complement cascade. Purified CR2 was examined for its cofactor activity in factor I-mediated cleavage of membrane-bound iC3b. CR2 plus C3b inactivator (I) released C3c from EA 125I-iC3b, and the release was inhibited when CR2 was preincubated with OKB7 monoclonal anti-CR2. Furthermore, immunoelectroblotting analysis showed crossreactivity of CR2 with 57H anti-CR1. These results indicate that CR2 has functional and antigenic similarity to CR1, thus providing a supporting evidence for placement of CR2 as a member of the recently defined gene family of C3- and C4-regulatory proteins composed of CR1, C4-binding protein, and factor H.

When pilus+ Gc were introduced into a male subject's urethra, they gave rise to pilus+ variants whose pilin mRNAs differed from that of input Gc. The differences stemmed from the Gc genome's single complete pilin gene having undergone gene conversion by different partial pilin genes' sequences and by different length stretches of a single partial pilin gene. In some instances, the variant's pilin mRNA appeared to reflect two independent gene-conversion events that used sequences from two different partial pilin genes. The resulting variants' pilins exhibited antigenic differences compared with the pilin polypeptide of input Gc; these differences were discernible by immunoblotting with mAbs. Amino acid and antigenic changes occurred in a segment of the variants' pilin polypeptides that previously was thought to be conserved or constant in sequence.

We screened a cDNA library prepared from a BALB.B10 CTL clone that expresses Qa-2 antigen, and isolated four clones derived from Q7b, a Qa region gene of C57BL/10. One of these Q7b cDNAs and the Q7b chromosomal gene were subcloned into expression vectors and transfected into L cells and R1.1 thymoma cells. We found that the chromosomal Q7b gene expresses Qa-2 on the surface of R1.1 cells, but not on L cells while the Q7b cDNA expresses protein on the surface of both cell types. The levels of Qa-2 expression do not correlate with the total levels of Q7b mRNA in these transfectants. Our results suggest that the tissue-specific expression of Qa-2 may be controlled, in part, by mechanisms of alternate RNA splicing. By using hybrid gene constructs, we have mapped the tissue-specific element to the 3' part of the gene, downstream of a site near the middle of exon 4. The hybrid polypeptides differ significantly in their transmembrane and cytoplasmic regions. These portions of the protein also may play a role in the tissue-specific expression of Qa-2.

C5-deficient mice differed from C5-sufficient mice both quantitatively and qualitatively in C5 protein, C5 mRNA, and the C5 gene. C5-deficient protein was present as decreased amounts of an unprocessed, single-chain precursor. C5-deficient mRNA was decreased in amount and present in two forms, the smaller of which was the same as the single form in normal cells. Nuclei from both normal and deficient cells contained the larger form of C5 mRNA, and C5-deficient DNA demonstrated differences from the normal pattern on Southern analysis for two restriction enzymes. These data suggest that the primary transcript of the C5-deficient gene is abnormal, retarding the processing of the C5 mRNA, and that the C5-deficient mRNA codes for an abnormal protein.

The cationic antibacterial proteins of rabbit PMN lysosomes have been resolved into at least five subfractions. Each of these showed substantial selectivity in its antibacterial action against several pathogenic bacteria, including two smooth and two rough Escherichia coli strains, three Staphylococcus aureus strains, one S. albus, three proteus species and four different cultures of streptococcus. Each of the subfractions possesses a different electrophoretic mobility. Amino acid analyses of the three most cationic components revealed high contents of arginine consistent with their relative electrophoretic mobilities and very high arginine to lysine ratios. Aromatic amino acids were present in very low concentrations in these proteins and their light absorption at 2800 A was correspondingly weak. The evidence of antibacterial specificity, along with marked differences in the arginine-lysine ratios, shows that the cationic antibacterial components of rabbit PMN lysosomes are biologically and chemically heterogeneous.