Biomedical subjects

K K Stanley

Publications and source records attributed to K K Stanley.

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PubMedDate not supplied

Domain structure, functional activity, and polymerization of trout complement protein C9.

The 3' region of trout C9 has been resequenced and found to differ from the previously published sequence (Stanley and Herz, EMBO J. 6:1951; 1987). In contrast to other sequenced C9 molecules, but in common with the other terminal complement components, trout C9 was found to contain an additional carboxy terminal thrombospondin domain. This domain does not restrict polymerization, as has been previously suggested (Stanley and Luzio, Nature 334:475; 1988), since alternative pathway activation of trout complement by rabbit erythrocytes lead to the formation of circular membrane attack complement lesions on the erythrocyte membrane. Although the trout C9 molecule is larger than human C9, the diameters of circular trout membrane attack complexes were approximately 30% smaller than their human counterparts. No lysis of erythrocytes bearing human C5b-7 or C5b-8 complexes was detected following incubation with trout serum containing EDTA, which suggests that trout C8 and C9 are unable to bind to human C7 and C8, respectively. Finally, trout and human serum were equally effective at killing the human serum-sensitive strain Salmonella minnesota Re595.

Amino Acid Sequence↗

PubMedDate not supplied

Horse complement protein C9: primary structure and cytotoxic activity.

Lack of hemolytic activity of horse serum is an inherent property of horse C9. To understand the molecular reasons for this deficiency we have cloned C9 cDNA from a horse liver cDNA library and have sequenced the cDNA yielding the complete coding sequence for horse C9. Purification of C9 from horse plasma and microsequencing established the N-terminus of the mature protein and verified that the correct horse C9 cDNA clone had been isolated. The deduced amino acid sequence corresponds to a mature protein of 526 amino acids that is 77% identical to human C9. It has the same domain structure as human C9 and contains 22 cysteines and four invariant tryptophans. The few differences include the N-terminus, which is an unblocked glycine in horse C9 but pyroglutamine in human C9, and three potential N-glycosylation sites compared to two in human C9. The N-terminal difference is unimportant since microsequencing of bovine C9, which is strongly hemolytic, established that it also has an unblocked glycine identical to horse C9. There are no obvious structural differences apparent that could resolve the differences in hemolytic potency between the two molecules. Aside from a few conservative replacements, both C9 sequences are identical between positions 250 and 360. This region includes the membrane interaction domain in C9 and the postulated transmembrane segment that is thought to constitute the wall of a putative transmembrane pore and, therefore, should be required for cytotoxicity. In agreement with this prediction we have observed that, in contrast to the marked decrease in hemolytic activity, horse C9 is very efficient in killing a variety of Gram-negative bacteria. These results demonstrate that horse C9 is a structurally competent molecule with efficient cytotoxic activity. Its inability to lyse erythrocytes may be related to the action of control proteins on target cell membranes.

Amino Acid Sequence↗

PubMedDate not supplied

Structural/functional similarity between proteins involved in complement- and cytotoxic T-lymphocyte-mediated cytolysis.

Cytolysis mediated by complement or cytolytic lymphocytes results in the formation of morphology similar lesions in the target membrane. These lesions, formed by the polymerization of C9 or perforin respectively, contribute the major killing action by causing osmotic lysis of the target cell. Following the suggestion of Mayer that the mechanisms of humoral and cell-mediated cytotoxicity might be related, studies into the morphology of the membrane lesions formed, and the proteins responsible for causing the lesions, have shown several similarities. While the lesion caused by natural and T-killer cells is a little larger than that caused by complement, its overall shape is similar and in both cases the cylindrical pore is formed by polymerization of a monomeric subunit, C9 (relative molecular mass, Mr = 71,000) for complement, and perforin (Mr = 66,000) for cell-mediated cytotoxicity. C9 has an absolute requirement for a receptor in the target membrane formed by the earlier membrane attack complex components, C5b, C6, C7 and C8 (ref. 8). For perforin, polymerization in a target membrane requires no receptor, specificity being derived from the specific recognition between killer and target cell. Both proteins can be made to polymerize in vitro by the addition of divalent cations (Zn2+ for C9 (ref. 16) and Ca2+ for perforin) and the resultant complexes closely resemble their physiological counterparts. Antibodies raised against lymphocyte-killed targets have also been shown to cross-react with complement proteins, but the antigenically related proteins were not determined in these studies. We show here using purified proteins that perforin, C9 and complexes involving C7 and C8 share a common antigenic determinant which is probably involved in polymerization.

Amino Acid Sequence↗

PubMedDate not supplied

Regulation of targeting signals in membrane proteins. [review].

Membrane proteins can contain short sequence motifs that determine their intracellular location, either by a retention or a retrieval mechanism. In both cases the targeting signal is essentially a specific binding site for other proteins that effect the localization. The folding of targeting motifs is often robust leading to a dominant effect in molecular cut and paste experiments designed to identify them. However regulation can also occur, allowing a single membrane protein to express different targeting signals at different locations in the cell. Regulation can be achieved by phosphorylation of the cytoplasmic domain leading to changes in binding affinity for effector proteins, or by masking of the targeting signal by complex formation.

Amino Acid Sequence↗