Complement C3: The Most Important Protein in the Complement System

• Gene Information: The human C3 gene is located on chromosome 19p13.3. It encodes complement component 3, the central effector protein of the complement cascade, belonging to the α2-macroglobulin superfamily of thioester-containing proteins.
• Protein Expression: C3 is predominantly synthesized and secreted by hepatocytes in the liver into peripheral blood. Smaller amounts are locally produced by renal tubular epithelial cells, macrophages and endothelial cells.
• Signaling Pathway: C3 serves as the universal convergence point for the classical, lectin and alternative complement pathways. All three cascades assemble C3 convertase to cleave intact C3 into two bioactive fragments: anaphylatoxin C3a and opsonin C3b. C3b further participates in C5 convertase formation to initiate terminal complement activation and MAC assembly, driving inflammatory response and tissue injury.
• Therapeutic Inhibition: These therapeutic candidates represent diverse strategies: small peptide inhibitors block the enzymatic cleavage of C3; siRNA molecules silence C3 expression; and antibodies directly neutralize C3 function. Their development underscores a multifaceted approach to curb pathological complement activation.

• The genome sequence of human C3 gene with R102G mutation encodes the whole molecule (ATG to STOP codon), including 3’UTR, were inserted into the Hipp11 (H11) locus .
• The human C3 expression is driven by the human C3 promoter.
• Exons 2-40 of the mouse C3 gene were knocked out. As a result, the mouse C3 protein is not expressed anymore.
• R102G was an SNP named C3F. The C3F variant is associated with several diseases, including IgA nephropathy, C3G, and age-related macular degeneration.

Strain specific analysis of C3 mRNA expression by RT-qPCR. The brain, liver, kidney, and eye tissues RNA were isolated from heterozygous B-hC3*R102G mice (H/+) (male and female n=3), then cDNA libraries were synthesized by reverse transcription, followed by RT-qPCR with human C3 primers. Human C3 is predominantly expressed in the liver. All results were normalized to the female eye C3 mRNA. Values are expressed as mean ± SEM.

Strain specific C3 expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hC3*R102G Mice plus by ELISA. Serum was collected from wild-type C57BL/6JNifdc mice (+/+), and homozygous B-hC3*R102G mice plus (H/H) and analyzed by ELISA. Mouse C3 was only detectable in wild-type C57BL/6JNifdc mice. Human C3 was exclusively detectable in homozygous B-hC3*R102G Mice plus. Values are expressed as mean ± SEM.

IHC Staining in B-H11-hC3*R102G, mC3 KO mice. The liver, kidney, and eye tissues of wild-type C57BL/6JNifdc mice (+/+) and homozygous B-H11-hC3*R102G, mC3 KO mice (H/H, -/-) were isolated at 16 weeks old and analyzed with IHC staining. C3 was detected in the liver, eye, and kidney of B-H11-hC3*R102G, mC3 KO mice and wild-type C57BL/6JNifdc mice, as the antibody cross-recognizes both human and mouse C3 (Abcam, ab200999). “+” indicate positive expression. Red arrows indicate a positive signal.

Histopathological analysis in B-hC3*R102G mice plus. The liver and kidney tissues of wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hC3*R102G mice plus (H/H) were isolated at 16 weeks old and analyzed with HE staining (male and female, n=6). The liver of male B-hC3*R102G mice plus (5/6) showed inflammatory cell infiltration. The kidneys of male (4/6) and female (5/6) B-hC3*R102G mice plus showed glomerular matrix proliferation. And there were no obvious abnormalities in wild-type C57BL/6JNifdc mice. Red arrows: inflammatory cell infiltration. Blue arrows: glomerular matrix proliferation.

Histopathological analysis in B-hC3*R102G mice plus. The kidneys of homozygous B-hC3*R102G mice plus (H/H) were isolated at 16 weeks old and 26 weeks old and analyzed with HE staining and PAS staining. The kidney of B-hC3*R102G mice plus showed glomerular cell proliferation, renal tubular basophilic changes, and dilation. Compared with wild-type mice, B-hC3*R102G mice plus exhibited increased PAS-positive staining indicative of glomerular mesangial matrix expansion. And there were no obvious abnormalities in wild-type C57BL/6JNifdc mice. Red arrows: Glomerular lesions, Blue arrows: Tubular lesions.

Analysis of blood biochemicals in B-hC3*R102G mice plus and wild-type C57BL/6JNifdc mice. Serum was collected from wild-type C57BL/6JNifdc mice (+/+) and and homozygous B-hC3*R102G mice plus (H/H) (male n=11, female n=16, 16 weeks old) and analyzed for biochemistry. The ALT, AST, and UREA were increased in male B-hC3*R102G mice plus. Values are expressed as mean ± SEM. Significance was determined by t-test, *p<0.05,**p<0.01, ***p<0.001.

Survival Curve of B-hC3*R102G mice plus. Graph showing the survival curve of homozygous B-hC3*R102G mice plus (H/H) (male, n=38). The survival rate of male B-hC3*R102G mice plus was around 50% at 16 weeks old. And we didn’t observe that mice died in the female B-hC3*R102G mice plus until 16 weeks old.