The present study provides evidence that the mechanism of PC deficiency caused by the A267T mutation  most likely involves impaired synthesis and also reduced secretion of the mutant. In contrast to what has been demonstrated for several other PC mutations causing PC deficiency [15, 20, 28], the effect of the present mutation on proteasomal degradation was minimal. However, like other PC mutations [7, 10, 13], the A267T variant was retained in the ER. Transient expression analysis demonstrated that the A267T PC protein levels secreted into the culture medium were seven and five fold less compared to WT PC in CHO-K1 and Huh7 cells, respectively. The intracellular levels of the A267T PC were also strongly reduced. No major differences in mRNA levels, half-lives or degradation were detected for the two PC variants. Immunofluorescence staining revealed that the mutant was observed mainly in ER while WT PC was detected both in ER and in Golgi indicating a rapidly processing and secretion of WT PC, while the majority of mutated PC most likely was retained in the cells in an attempt to be folded correctly.
PC deficiency is an inherited disorder associated with a high risk of recurrent venous thrombosis [2, 3]. In vitro studies of various mutations in the PROC gene have provided insight into how these mutations can cause PC deficiency [7–15, 20, 28]. Most of these studies revealed that the PC deficiency was due to impaired secretion caused by intracellular degradation of the mutated proteins [8, 10, 11, 15, 20, 28]. Enhanced proteolytic degradation of mutated proteins is a common molecular pathological mechanism in many diseases . Degradation is often associated with impaired intracellular transport of proteins in the producing cells . Intracellular protein transport through the secretory pathway in eukaryotic cells depends on a proper folding of proteins in ER [17, 18]. Several transfection studies have described impaired transport of proteins from ER to Golgi due to prolonged binding of the mutated proteins with chaperones which facilitate a folding process in ER [7, 32]. Mutated/misfolded proteins are frequently subjected to subsequent degradation by quality control mechanisms involving ERAD . Not all mutated proteins will be recognized as misfolded by the ER quality control system. Some of them can remain in ER for an extended time period without being sent for disposal in ERAD [24, 33–35]. It can be speculated whether the effect a mutation has on 3 D structure of a protein can determine which mechanism that will be chosen in each particular case. It is likely that mutations, which are able to cause severe conformational changes, trigger ERAD to become hyperactive resulting in nearly complete degradation of the mutated proteins whereas a mutation with minor effects on the protein structure can result in several rounds of the folding process and thereby increased ER retention and attenuated degradation. The latest database on 3D-structure of PC http://www.itb.cnr.it/procmd/, obtained by computational approaches, interpreted the A267T mutation as not being disruptive for the structure and function of PC. In addition, alignment of the PC amino acid sequence and other evolutionary related human vitamin-K-dependent factors revealed that the A267 is not strictly conserved. All these facts may indicate that the investigated mutation leads to only minor effects on the PC molecule and inefficient ERAD degradation.
The fact that the intracellular level of A267T PC was reduced and the mutated PC was not degraded faster compared to WT PC could indicate that mRNA translation might be affected as indicated by other reports [32, 36]. The most immediate response to ER stress, which can occur as a result of misfolded protein accumulation in ER, is transient attenuation of mRNA translation which prevents influx of newly synthesized polypeptides in ER [16, 23]. Although no evidence of ER stress is presently available, one might speculate that reduced mRNA translation of mutated PC as a result of UPR could explain the fact that the intracellular levels of A267T PC was reduced compared to the WT PC levels. In-vitro translation experiments might have revealed if this was the case. Since the effect of inhibition of proteasomal degradation in this study was found to be insufficient to explain the reduced intracellular and secreted levels of A267T PC, the involvement of additional degradation pathways can not be ruled out. A small number of mutated secretory proteins have been reported to be degraded both by proteasomes and other cytosolic proteases (often serine/cysteine) simultaneously [37–39].
In some of the previous studies where the proteasomal degradation was found to be the dominant mechanism of PC deficiency [15, 20, 28], it was shown that this degradation was independent of ubiquitinylation of mutated PC. Ubiquitinylation is essential in several cellular processes including targeting proteins for proteasomal degradation and is catalyzed by the sequential action of various enzymes . In our study, we found that inhibition of ubiquitinylation by pyrazone (PYR-41) had no effect on the PC levels of either WT or A267T PC (data not shown) and indicated that proteasomal degradation of the investigated PC variants was independent of ubiquitin.
Intracellular protein transport through the secretory pathway in eukaryotic cells consists of translocations from ER to Golgi and from Golgi to secretory vesicles . Like other secretory glycoproteins, PC undergoes post-translational processing such as glycosylation, γ-carboxylation and β-hydroxylation. N-linked glycosylation of PC takes place before transport to the Golgi apparatus and possibly as a co-translational event in ER. On their way through the Golgi, the carbohydrate side chains of PC undergo further modifications which lead to resistance against Endo-H treatment of the protein in Golgi . The Endo-H digestion analysis in the present study revealed that both WT and A267T PC proteins in the cell lysates were sensitive to the Endo-H treatment, indicating that the PC was localized predominantly to the ER. However, the immunofluorescence analysis in this study demonstrated that a fraction of WT PC was located in Golgi, and thus, we expected some of the cellular WT PC to be resistant to Endo-H. This was however, not the case. In agreement with other studies [10, 15] we therefore assume that most WT PC resided in the ER and was secreted quickly subsequent to post-translational modifications.