Distinction between processing of normal and mutant complement C3 within human skin fibroblasts

Abstract

Inherited C3 deficiency may result from mutations in the C3 gene affecting transcription or translation (type I deficiency). We described a type II C3 deficiency caused by a mutation yielding an abnormal non-secreted C3. The post-translational processing of mutant and normal C3 was analyzed in fibroblasts grown from skin biopsies. Mutant C3 is located mainly in the endoplasmic reticulum (ER), whereas normal C3 is seen evenly distributed throughout the cytoplasm. Most of the mutant C3 is degraded within the cell, and only a small fraction (around 8%) is secreted after 20 h chase. Processing of C3 at 19 degrees C was reduced in normal fibroblasts but completely blocked in mutant fibroblasts. ATP depletion blocked processing of normal proC3 to C3. In contrast, the mutant proC3 was partly degraded in ATP-depleted cells, yet its complete degradation and secretion were blocked. Intracellular degradation of the mutant C3 was not inhibited by NH4Cl, thus excluding cleavage within lysosomes. These results demonstrate that the type II mutant C3 studied here is retained in the ER probably by a quality contol machinery that identifies abnormal protein folding. Consequently, it is destined to undergo a two-step intracellular degradation; an initial ATP-independent step followed by an ATP-dependent step.

Hereditary Human Complement C3 Deficiency Owing to Reduced Levels of C3 mRNA

“…The molecular defects leading to the primary C3 deficiency are heterogeneous. In previously investigated cases, these included partial deletions [19], point mutations [20–22] and unidentified defects leading to diminished C3 production [23] or secretion [24,25]. Southern blot analysis of genomic DNA from the proband, his mother and normal individuals digested with restriction endonucleases and hybridized with a full length C3 cDNA probe, confirmed that this deficiency did not arise from large genomic deletions, insertions or rearrangements in the C3 gene (data not shown).…”

Intracellular complement activation—An alarm raising mechanism?

“…Sequence analysis of two of our C3deficient siblings showed a deletion mutation in the C3 beta chain (unpublished). This and similar mutations could potentially lead to a failure in correctly folded C3, and thus inhibit secretion, while simultaneously producing a C3a-containing C3 remnant, which would fulfill the intracellular function [52,127,129]. Whether the C3a produced by the peripheral blood mononuclear cells from C3-deficient patients maintains the ability to stimulate neutrophil oxidative burst, as described for the cathepsin L generated C3a, remains to be determined [52].…”

“…This is an intriguing observation, given that C3-deficient patients do have surviving and proliferating T-cells in circulation. A closer investigation of these patients revealed that in a number of cases C3-serum deficiency did not correlate with complete C3 deficiency [125][126][127][128][129]. Thus C3-products can be observed in isolated cells from apparently C3-deficient patients, and gene expression profiling has revealed C3 mRNA expression in peripheral blood mononuclear cells [52,116].…”

Deficiências concomitantes da proteína reguladora Fator H e do componente C9 do complemento.

“…A metodologia empregada neste experimento foi adaptada a partir da publicada por Fishelson et al (1999).…”