N-deglycosylation of human complement component C9 reduces its hemolytic activity

Area of Science:

• Immunology
• Glycobiology
• Biochemistry

Background:

• Human complement component C9 (C9) is a key protein in the complement system.
• The role of glycosylation in C9's hemolytic activity is not fully understood.

Purpose of the Study:

• To investigate the impact of enzymatic deglycosylation on the hemolytic activity of human C9.
• To determine the specific role of N-linked and O-linked carbohydrates in C9 function.

Main Methods:

• Enzymatic deglycosylation of native C9 using glycopeptidase F (PNGase F) and neuraminidase.
• Analysis of molecular weight changes and residual carbohydrate content.
• Assessment of hemolytic activity (CH50) before and after deglycosylation.

Main Results:

• PNGase F treatment stepwise reduced C9's molecular weight, indicating two N-linked carbohydrate chains.
• Removal of 88% of N-linked oligosaccharides led to an 80% reduction in hemolytic activity.
• N-deglycosylated C9 retained O-linked glycans, suggesting their lesser role in hemolysis.

Conclusions:

• N-linked carbohydrates on human C9 are essential for its full hemolytic activity.
• Unlike other glycoproteins, deglycosylation significantly impacts C9's functional capacity.