A simple alternative pathway for hemolytic assay of human complement component C3 using methylamine-treated plasma

The cleavage of human complement component C5 to fragment C5b by the alternative pathway C5 convertase was studied. The alternative-pathway C5 convertase on zymosan can be represented by the empirical formula zymosan--C3b2BbP. Both properdin-stabilized C3 and C5 convertase activities decay with a half life of 34 min correlating with the loss of the Bb subunit. The C5 convertase functions in a stepwise fashion: first, C5 binds to C3b and this is followed by cleavage of C5 to C5b. The capacity to bind C3b is a stable feature of component C5, as C5b also has this binding capacity. Component C5, unlike component C3, does not form covalent bonds with zymosan after activation, and C5 is not inhibited by amines. Therefore C5, although similar in structure to C3, does not appear to contain the internal thioester group reported for C3 and C4.

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Amino acid sequence analysis of the glycopeptides from human complement component C3

FEBS Letters ◽

10.1016/0014-5793(86)80649-4 ◽

1986 ◽

Vol 202 (1) ◽

pp. 59-62 ◽

Cited By ~ 10

Author(s):

Karen G. Welinder ◽

Allan Svendsen

Keyword(s):

Amino Acid ◽

Sequence Analysis ◽

Amino Acid Sequence ◽

Complement Component ◽

Human Complement ◽

Amino Acid Sequence Analysis ◽

Complement Component C3 ◽

Human Complement Component

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Mycobacterial Protein HbhA Binds Human Complement Component C3

Infection and Immunity ◽

10.1128/iai.69.12.7501-7511.2001 ◽

2001 ◽

Vol 69 (12) ◽

pp. 7501-7511 ◽

Cited By ~ 34

Author(s):

Stacey L. Mueller-Ortiz ◽

Audrey R. Wanger ◽

Steven J. Norris

Keyword(s):

Amino Acid ◽

Cell Membrane ◽

Binding Protein ◽

Complement Component ◽

Mononuclear Phagocytes ◽

Full Length ◽

Dependent Manner ◽

Human Complement ◽

Complement Component C3 ◽

Human Complement Component

ABSTRACT Mycobacterium tuberculosis and Mycobacterium avium are facultative intracellular pathogens that are able to survive and replicate in mononuclear phagocytes. Human complement component C3 has previously been shown to mediate attachment and phagocytosis of these bacteria by mononuclear phagocytes. In this study, a C3 ligand affinity blot protocol was used to identify a 30-kDa C3-binding protein in M. tuberculosis andMycobacterium smegmatis and a 31-kDa C3-binding protein inM. avium. The C3-binding proteins in M. tuberculosis and M. avium localized to the cell membrane fraction and partitioned to the detergent fraction during Triton X-114 phase partitioning. The C3-binding protein from M. tuberculosis was partially purified using a cation exchange column and was shown to bind concanavalin A. The N terminus and an internal fragment of the partially purified C3-binding protein were subjected to amino acid sequence analysis. The resulting amino acid sequences matched the M. tuberculosis heparin-binding hemagglutinin (HbhA) protein. Recombinant full-length HbhA and the C terminus of HbhA fused to maltose-binding protein, but not recombinant HbhA lacking the C-terminal region, bound human C3. Recombinant full-length HbhA coated on polystyrene beads, was found to enhance the adherence and/or phagocytosis of the coated beads to J774.A1 cells in both the presence and absence of human serum. The presence of complement-sufficient serum increased the adherence of the HbhA-coated beads to the J774.A1 cells in a C3-dependent manner. If HbhA within the bacterial cell membrane functions similarly to isolated HbhA, this protein may enhance the adherence and phagocytosis of M. tuberculosis and M. avium to mononuclear phagocytes through the binding of C3 and interaction with C3 receptors on mononuclear phagocytes.

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