Topology of the membrane-bound form of complement protein C9 probed by glycosylation mapping, anti-peptide antibody binding, and disulfide modification

Abstract

The two N-linked oligosaccharides in native human C9 were deleted by site-specific mutagenesis. This aglycosyl-C9 did not differ from its native form in hemolytic and bactericidal activity. A new N-glycosylation site (K311N/E313T) was introduced into the turn of a helix-turn-helix [HTH] fold that had been postulated to form a transmembrane hairpin in membrane-bound C9. This glycosylated form of human C9 was as active as the native protein suggesting that the glycan chain remains on the external side of the membrane and that translocation of this hairpin is not required for membrane anchoring. Furthermore, flow cytometry provided evidence for the recognition of membrane-bound C9 on complement-lysed ghosts by an antibody specific for the HTH fold. A new N-glycosylation site (P26N) was also introduced close to the N-terminus of C9 to test whether this region was involved in C9 polymerization, which is thought to be required for cytolytic activity of C9. Again, this glycosylated C9 was as active as native C9 and could be induced to polymerize by heating or incubation with metal ions. The two C-terminal cystines within the MACPF domain could be eliminated partially or completely without affecting the hemolytic activity. Free sulfhydryl groups of unpaired cysteines in such C9 mutants are blocked since they could not be modified with SH-specific reagents. These results are discussed with respect to a recently proposed model that, on the basis of the MACPF structure in C8alpha, envisions membrane insertion of C9 to resemble the mechanism by which cholesterol-dependent cytolysins enter a membrane.

Figure 1

C9 glycan mutants and location of the respective glycosylation sites. Tag refers to the addition of a C-terminal hexahistidine sequence that was not removed from the individual proteins. Secretion indicates whether the proteins were secreted by COS-7 and also by Sf9 cells.

Figure 2

Putative TMH sequences in human C8 and C9 (Hadders et al., 2007). Dotted lines indicate disulfide bonds.

Figure 3

Hemolytic activity and sulfhydryl labeling of C9-cysteine mutants. C9 mutant proteins with modified cystine residues were expressed in COS-7 and Sf9 cells and tested for secretion into the culture fluid, hemolytic activity, and labeling by SH-specific fluorescent probes. All secreted proteins have a C-terminal His6 tag allowing isolation of the secreted proteins by affinity chromatography. Protein concentrations were determined by immunoblotting and equivalent amounts of proteins were used to determine hemolytic activities which were not significantly different from recombinant wild type protein. SH-specific labeling of C9 proteins was attempted using a variety of different reagents (listed in the Experimental Procedures section) with bovine serum albumin (BSA) serving as a control. Low level labeling of C9 proteins was about 20% of that obtained with BSA.

Figure 4

Functional activity of C9 glycan mutants. Panel A: Hemolytic activity of C9 mutants expressed in COS-7 and Sf9 cells a: Serum C9 b: rC9 expressed in COS-7 cells c: C9-T258M (pYW30) expressed in COS-7 cells d: C9-T396M (pYW31) expressed in COS-7 cells e: C9-T258M/T396M (pYW33) expressed in Sf9 cells f: C9-258M/396M – K311N/E313T (pYW34) expressed in Sf9 cells g: C9-258M/396M – P26N (pVR11) expressed in Sf9 cells Standard assays of C9 hemolytic activity (Sodetz and Esser, 1988) consisted of 5×10⁸ sheep EAC1-8 and 50 μL of sample dilution in a total volume of 0.1 mL isotonic buffer. Incubations were conducted for 45 min at 37°C followed by addition of ice-cold buffer and centrifugation. 100% lysis was achieved by addition of 1 mL H₂O in place of buffer after incubation. The amount of recombinant C9 used in all assays was approximately 5 ng/mL as measured by ELISA (Tomlinson et al., 1993). Panel B: Killing of E. coli strain C600 by (a) C9-deficient serum reconstituted with 8 μg of rC9 or (b) with 8 μg aglycosyl-C9 (C9-258M/396M) per mL. (c) C9-deficient human serum. Error bars indicate means ± standard deviations (from 3-5 experiments).

Figure 5

Lectin blotting of C9 and glycan mutant proteins after SDS-Page under non-reducing conditions. Top Panel: Detection by Coomassie blue (CB) staining and Sambucus nigra agglutinin (SNA) blotting. Lane 1: 5 μg serum C9 Lane 2: 0.5 μg serum C9 Lanes 3 & 5: Fetuin (positive control) Lane 4: Carboxpetidase Y (negative control) Lane 6: C9-258M/396M- K311N/E313T expressed in COS-7 cells Bottom Panel: Detection by Coomassie blue (CB) staining and Galanthus nivalis agglutinin (GNA) blotting Lane 1: 5 μg serum C9 Lane 2: rC9 expressed in Sf9 cells Lane 3: C9-258M/396M- P26N expressed in Sf9 cells Lane 4: Carboxpetidase Y (positive control) Lane 5: rC9 expressed in COS-7 cells

Figure 6

Recognition of membrane-bound C9 by antipeptide antibody binding as measured by flow cytometry. The left trace (white peak in both panels) shows the autofluorescence of complement-lysed ghosts incubated with non-immune IgG. Incubation with anti-(305-319) IgG (top panel) or anti-(311-324) (bottom panel) shifts the fluorescence (middle trace, gray peak in both panels) to the right and maximum fluorescence is obtained after incubation with polyclonal anti-C9 (black peak in both panels).

Figure 7

Comparison of the first putative transmembrane hairpin (TMH1) sequence in C9 from different species. The cartoon shows locations of two β-strands within the putative transmembrane hairpin regions. Amino acids are coded according to character: hydrophilic (white letters in gray box), hydrophobic (black letters) and those that do not fit the alternating hydrophilic/hydrophobic amphipathic pattern are shown in underlined bold black letters. Potential glycosylation sites [NXS/T] are placed within rectangles.

Figure 8

Membrane anchoring of C9. Panel A: Transmembrane orientation of C9 as proposed by Peitsch et al. (1990). Panel B: Monotopic anchoring of C9. The C9 sequence shown is the same in both panels and is homologous with helices D and E in the MACPF C8α-γ structure of Slade et al. (2008).