The Carboxyl-Terminal Region of Protein C Is Essential for Its Secretion

Abstract

We have previously reported a mutated protein C, designated protein C Nagoya (PCN), characterized by the deletion of a single guanine residue (8857G). This frameshift mutation results in the replacement of the carboxyl-terminal 39 amino acids of wild-type protein C (G381-P419) by 81 abnormal amino acids. This elongated mutant was not effectively secreted, and was retained in the endoplasmic reticulum. To determine why PCN is not secreted, we constructed a series of mutants from which some or all of the 81 amino acids were deleted. None of these shortened proteins were secreted from producing cells, indicating that the carboxyl-terminal extension is not mainly responsible for the intracellular retention of PCN, and that the 39 carboxyl-terminal amino acids of wild-type protein C are required for secretion. To determine which residues are essential for the secretion of protein C, deletion mutants of the carboxyl-terminal region (D401-P419) were prepared. Metabolic labeling showed that mutants of protein C truncated before W417, Q414, E411, or K410 were efficiently secreted. On the other hand, the mutants truncated before D409 were retained and degraded intracellularly. Immunofluorescence and immunoelectron microscopy showed that truncation before D409 blocks the movement from rough endoplasmic reticulum to the Golgi apparatus. To understand the conformational change in the carboxyl-terminal region, two models of truncated activated protein C were constructed using energy optimization and molecular dynamics with water molecules.

The carboxyl‐terminal region is NOT essential for secreted and functional levels of coagulation factor X

“…The study rationale stems from previous observations showing that the natural full‐length carboxyl‐terminus is essential for the secretion of FVII, FIX and PC .…”

“…Interestingly, a large difference in secretion levels was observed between FX variants truncated at residues 468 and 467, a finding comparable to that obtained for PC and marking the carboxyl‐terminal region of chymotrypsin (Fig. ).…”

“…One of the protein regions displaying the most striking differences is represented by the carboxyl‐terminus, which varies both in length and amino acid composition. Previous studies conducted by us and others , through the characterization of nonsense and missense as well as frame‐shifted variants of FVII, FIX and PC, have demonstrated that this region represents a key determinant for efficient secretion. So far, very little is known about FX, which is characterized by the most extended carboxyl‐terminal region.…”

Replacement of the Y450 (c234) phenyl ring in the carboxyl‐terminal region of coagulation factor IX causes pleiotropic effects on secretion and enzyme activity

“…1), which could underlie different structural and/or functional roles. Previous studies indicated that the carboxyl‐terminus has an essential role for the secretion of FIX [11] and also of FVII [12] and PC [13]. On the other hand, alterations in this region resulted in poor secretion of normal or hyperactive variants of PC and FVII [14].…”

Identification of protein Sα gene mutations including four novel mutations in eight unrelated patients with protein S deficiency

“…In this study, we demonstrated that the exon 10 +5A→G mutation also occurred in an apparently distinct ethnic population, suggesting that this high frequent mutation could be due to the presence of a mutational hot spot. The other 891G→T mutation converts Glu208 to a stop codon, supposing that a mutant RNA would be degraded intracellularly (Yamazaki et al , 1995) or that a non‐functional aberrant protein might be translated from the residual mutant RNA and degraded intracellularly because of abnormal protein conformation, as found in other coagulation proteins such as protein C (Katsumi et al , 1998).…”