Two novel mutations in EGF‐like domains of human factor IX dramatically impair intracellular processing and secretion

Abstract

We investigated the mechanisms responsible for severe factor IX (FIX) deficiency in two cross-reacting material (CRM)-negative hemophilia B patients with a mutation in the first and second epidermal growth factor (EGF) domains of FIX (C71Y and C109Y, respectively). We have determined the kinetics of mutant FIX biosynthesis and secretion in comparison with wild-type FIX (FIXwt). In transfected cells, FIXwt was retrieved as two intracellular molecular forms, rapidly secreted into the culture medium. One appeared to be correctly N-glycosylated, and corresponded to a form trafficking between the endoplasmic reticulum (ER) and Golgi apparatus. The other corresponded to the mature form, ready to be secreted, exhibiting correct N-glycosylation and sialylation. In contrast, the two mutants, FIXC71Y and FIXC109Y, were not secreted from the cells and did not accumulate intracellularly. Relative to FIXwt, they were retained longer in the ER and were only N-glycosylated. In addition, the intracellular concentration of the FIX mutants increased when ALLN, an inhibitor of cysteine proteases and of the proteasome degradation pathway, was added to the culture medium. Both the FIX mutants and FIXwt were associated in the ER with the 78-kDa glucose-regulated protein (GRP78/BiP) and calreticulin (CRT), though the amount of CRT associated with the two mutants was twice as strong as with FIXwt. These results strongly suggest that chaperone and lectin molecules act in concert to ensure both proper folding of FIXwt and the retention of mutant molecules.

Single synonymous mutation in factor IX alters protein properties and underlies haemophilia B

“…Non-synonymous mutations (altering protein structure) in the second EGF-like domain (where Val107Val mutation is located) were shown to cause impaired intracellular processing and secretion 26. Current knowledge about the effects of synonymous mutations on protein function suggests that they can also alter protein structure.…”

“…These are associated with subtle changes in its structure caused by altered translation dynamics (figure 3). Note that non-synonymous mutations in the second EGF-like domain were previously shown to cause FIX impaired intracellular processing and secretion 26. We have previously presented an extensive review of the multiple mechanisms by which synonymous mutations affect protein structure and function and the methods that may be used to study these 4.…”

Maternal low-level somatic mosaicism of Cys155Tyr of F9 in severe hemophilia B

“…The disruption of the Cys141 (Cys95)-Cys155 (Cys109) disulfide bond could lead to a FIX activity less than 1% [6]. A previous study showed that mutant FIX carrying Cys155Tyr (Cys109Tyr) was not secreted from the cells and did not accumulate intracellularly [18].…”

Therapeutic plasma proteins – application of proteomics in process optimization, validation, and analysis of the final product

“…Other changes, e.g. in the epidermal growth factor-like domain, can negatively influence secretion of this glycoprotein [122,123]. On the other hand, targeted changes and modifications to this clotting factor can significantly improve its activity and prolong its lifetime in the bloodstream.…”