Biomedical subjects

E C Petrella

Publications and source records attributed to E C Petrella.

4 recordsLinked to original sources

PubMed1992

Primary structure of cobra complement component C3.

Complement component C3 is a multifunctional protein known to interact specifically with more than 10 different plasma proteins or cell surface receptors. Cobra venom contains cobra venom factor, a structural analogue of C3 that shares some properties with C3 (e.g., formation of a C3/C5 convertase) but differs in others (e.g., susceptibility to regulation by factors H and I). The elucidation of structural differences between C3 and cobra venom factor can be expected to help identify functionally important regions of C3 molecules. To that end we have undertaken the molecular cloning of both cobra C3 and cobra venom factor to take advantage of the unique biologic system where both proteins are produced by the same species. We report the primary structure of cobra C3 mRNA and the derived protein structure. Cobra C3 mRNA is 5211 bp in length. It contains an open reading frame of 4953 bp coding for a single pre-pro-C3 molecule, consisting of a 22-amino acid signal sequence, a 633-amino acid beta-chain (70 kDa), and a 992-amino acid alpha-chain (112 kDa) which is separated from the beta-chain by four arginine residues. There are no N-glycosylation sites in cobra C3. Cobra C3 exhibits approximately 58% nucleotide sequence identity with C3 from mammalian species. At the protein level, sequence identity is approximately 52% and sequence similarity approximately 71%. All 27 cysteine residues are highly conserved as are the C3 convertase cleavage site, the thioester site, and the factor B binding site. Cobra C3 also seems to have homologous binding sites for factor H and properdin, as well as a conserved sequence in the functionally important region of the C3a anaphylatoxin. The sequence homology at the CR2 and CR3 binding sites does not exceed the overall sequence homology. Accordingly, the existence of CR2 and CR3 binding sites can neither be deduced nor excluded.

Amino Acid Sequence↗

PubMed1990

C3-cleaving membrane proteinase. A new complement regulatory protein of human melanoma cells.

Human melanoma cells resistant to killing by the R24 mAb and human complement rapidly degrade surface-deposited C3b (M. Panneerselvam, S. Welt, L. J. Old, C.-W. Vogel. 1986. J. Immunol. 136:2534). We report that C-resistant melanoma cells express a membrane proteinase that can cleave C3b, generating a cleavage product with a molecular mass of approximately 30 kDa. The C3-cleaving proteinase was identified on the melanoma cells by its cross-reaction with antiserum to p57, a C3-cleaving proteinase previously isolated from human E membranes (C. Charriaut-Marlangue, M. Barel, R. Frade. 1986. Biochem. Biophys. Res. Commun. 140:1113). Preincubation of the C-resistant melanoma cells with anti-p57 IgG or their F(ab')2 fragments increased their susceptibility to complement killing from 25% to approximately 50% and reduced the rate of C3b cleavage and the amount of the 30-kDa fragment generated on the cells. Anti-p57 IgG stained C-resistant melanoma cells by indirect immunofluorescence and precipitated a protein with an apparent molecular mass of 65 kDa. This membrane protein, termed p65, was not detectable on C-susceptible melanoma cells. Membrane extracts from C-resistant melanoma cells also showed C3-cleaving activity when incubated with purified C3 or C3b, similarly generating a C3 fragment of approximately 35 kDa. This fluid-phase C3 cleaving activity could be partially inhibited by anti-p57 IgG. These data suggest that p65 is a C3-cleaving proteinase, antigenically related to p57, that is expressed on C-resistant melanoma cells and responsible for the C resistance of these cells. We propose that the membrane-bound C3-cleaving proteinase represents another C regulatory protein protecting host cells against killing by C.

Blotting, Western↗

PubMed1990

Complement killing of human neuroblastoma cells: a cytotoxic monoclonal antibody and its F(ab')2-cobra venom factor conjugate are equally cytotoxic.

Only a few monoclonal antibodies mediate complement lysis of tumor cells, but for several antibodies it has been demonstrated that a complement-activating function can be introduced by covalent coupling of cobra venom factor (CVF), a non-toxic glycoprotein which is a structural and functional homologue of human complement component C3. In this study we compared the efficacy of complement killing of human neuroblastoma cells by the complement-activating monoclonal antibody 3F8 directed against the GD2 ganglioside antigen with that of its F(ab')2-CVF conjugate. At equal numbers bound per cell the 3F8 antibody and the 3F8 F(ab')2-CVF conjugate were found to be equally cytotoxic in the presence of complement from several species including human. Maximal killing reached up to 98%. The kinetics of killing and the bivalent metal requirement confirmed that the cytotoxic activity of the 3F8 antibody is mediated via the classical pathway and that of the 3F8 F(ab')2-CVF conjugate via the alternative pathway. To achieve a comparable degree of killing, an approximately eight-fold higher concentration of the 3F8 F(ab')2-CVF conjugate was required which appears to be a consequence of the approximately eight-fold lower binding activity of the 3F8 F(ab')2-CVF conjugate compared to the intact 3F8 antibody. Our data suggest that the coupling of CVF to non-cytotoxic antibodies allows the generation of conjugates with a cytotoxic activity similar to that of inherently cytotoxic antibodies.

Antibodies, Monoclonal↗

PubMed1987

Antibody conjugates with cobra venom factor. Synthesis and biochemical characterization.

Immunoconjugates are semi-synthetic hybrid proteins which bear great promise to become a new generation of anti-tumor agents. While many immunoconjugates have been shown to be selectively cytotoxic in in vitro model systems, dramatic in vivo anti-tumor effects have not been reported. To improve the activity of immunoconjugates, careful structure-function analyses have to be performed. We report here such an analysis for immunoconjugates consisting of a monoclonal anti-tumor antibody (MoAb) and cobra venom factor (CVF), the complement-activating glycoprotein from cobra venom, synthesized with the heterobifunctional crosslinking reagent N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). It is shown that a reaction mixture after protein coupling contains free MoAb and CVF as well as hybrid proteins of different compositions (dimers (MoAb-CVF), trimers (MoAb2-CVF, MoAb-CVF2), tetramers (MoAb-CVF3, MoAb2-CVF2, MoAb3-CVF), and some higher oligomers). While free MoAb and CVF can be removed by size exclusion chromatography, separation of different oligomeric hybrid proteins is not possible by this method. From the biochemical characterization of the hybrid proteins, which included the determination of sedimentation coefficients, recording of circular dichroism spectra with subsequent determination of secondary structure, and ultrastructural analysis by transmission electron microscopy, it was concluded that the two proteins do not undergo major structural changes upon coupling, and that the coupling of the two proteins is random with no preferential relative orientation. The functional inactivation of CVF was substantial (approximately 70%) due to both derivatization with SPDP and subsequent conjugation to the MoAb, with conjugation being relatively more inactivating than derivatization. In contrast, the binding activity of the antibody was far less susceptible to inactivation. In conclusion, our data indicate that immunoconjugate synthesis with heterobifunctional crosslinking reagents results in a mixture of heterogeneous hybrid proteins and causes substantial functional inactivation. For successful in vivo anti-tumor activity of future immunoconjugates with CVF and other protein ligands better methods for immunoconjugate synthesis will have to be developed.

Antibodies, Neoplasm↗