Altered glycosylation and selected mutation in recombinant human complement component C9: effects on haemolytic...

Area of Science:

• Molecular Biology
• Immunology
• Protein Engineering

Background:

• The complement system is crucial for innate and adaptive immunity.
• Complement component C9 is the terminal protein in the complement cascade, forming the membrane attack complex.
• Understanding C9 structure-function relationships requires reliable recombinant protein production.

Purpose of the Study:

• To synthesize recombinant wild-type and mutated human complement component C9 (rC9) using a baculovirus expression system.
• To characterize the functional properties of rC9 and compare them to native C9.
• To investigate the impact of specific mutations on C9 activity and secretion.

Main Methods:

• Baculovirus-infected insect cells (Spodoptera frugiperda and Trichoplusia ni) were used for recombinant protein expression.
• Native signal peptide and insect cell lines were modified to optimize rC9 yield.
• Functional assays included hemolytic activity, enzymatic digestion, antibody binding, deglycosylation, and Zn2+-induced polymerization.

Main Results:

• Wild-type rC9 exhibited indistinguishable hemolytic activity, digestion patterns, antibody recognition, deglycosylation profile, and Zn2+-induced polymerization compared to native C9.
• Optimized expression yielded 5 µg/ml of rC9 supernatant.
• A mutant with altered putative calcium-binding sites retained wild-type activity, while an N-terminal deletion mutant showed secretion-induced inactivation, and a cysteine mutant exhibited significantly reduced secretion.

Conclusions:

• The baculovirus expression system can produce functional wild-type human C9.
• Specific mutations differentially affect C9 secretion and activity, providing insights into its functional domains.
• This recombinant C9 system is valuable for future studies on complement C9 structure and function.