Eukaryotic protein homeostasis, termed proteostasis, is maintained by a network of pathways controlling protein synthesis, folding, trafficking, aggregation, disaggregation, and degradation . In particular, the proteostasis network is involved in maintaining the dynamic interplay between folding and export of proteins . Secretion is a fundamental function of every cell. Current knowledge on protein secretion originates ∼30 years ago by the work of Palade and colleagues . This work first established the vesicle transport hypothesis, which states that the transfer of cargo molecules between organelles of secretory pathway is mediated by shuttling transport vesicles . Accordingly to this theory, newly synthesized secretory proteins pass through a series of membrane-enclosed organelles including the endoplasmic reticulum (ER), the Golgi complex, and secretory granules, before they reach the extracellular space. Even proteins destined for residence at the plasma membrane, endosomes, or lysosomes share the early steps of this pathway (i.e., the ER and the Golgi complex) with secreted proteins. The eukaryotic secretory pathway is responsible for delivery of a wide number of proteins to their specific cell location. Secretory proteins contain sorting elements that are recognized by the intracellular transport machinery at multiple steps of the way from synthesis to specific location . Proper selection of appropriate proteins for incorporation into nascent vesicles is crucial for secretory pathway homeostasis . Coat protein complexes are major components of this machinery. Three well-characterized coat complexes, clathrin, and coat protein complexes I and II (COPI and COPII), have been described; they are multisubunit complexes that recognize specific protein sorting signals and they selectively sort proteins into carrier vesicles.
This review describes fundamental principles as well as contemporary aspects of protein intracellular trafficking providing new insights that have emerged in the field of hematology diseases in recent years. Since protein intracellular pathway has been extensively covered by excellent reviews [4, 6], it will not be covered in details in this review.
In this review we will describe human genetic disorders associated with defects in COPII machinery. In particular, we will focus on two COPII-related genetic disorders: the Congenital Dyserythropoietic Anemia type II (CDA II) and the Combined Deficiency of Factor V and VIII (F5F8D).
Trafficking pathway ER–Golgi
Transportation can be divided into four main phases: vesicle budding, the selection of proteins to transport, addressing, and the fusion of the vesicle membrane with the target. The vesicle bud by a membrane called the donor, which also allows the selective incorporation of cargo proteins in the vesicle and retain residents in the donor compartment. These vesicles are directed to specific target compartments where they can deliver their loads after fusion with the membrane . The export of proteins from the ER has been well defined in yeast, both S. cerevisiae  and P. pastoris , and in mammalian cells . Figure 1 shows a diagrammatic representation of this pathway: nascent secretory proteins are translated and folded at the ER and then packaged into COPII vesicles for anterograde transport to pre-Golgi and Golgi compartments. Forward transport of folded secretory proteins in COPII vesicles is also balanced by a retrograde transport pathway that relies on COPI to recycle vesicle components and retrieve escaped ER resident proteins (Fig. 1). While in S. cerevisiae vesicle budding appears to occur stochastically across the entire ER membrane, in P. pastoris and in mammalian cells, this event is highly organized, occurring at discrete sites called transitional ER (tER) or ER exit sites (ERES) [3, 9-11]. These sites are relatively immobile structures and face out towards assemblies of ERGIC clusters (ER-Golgi intermediate compartment) [12, 13]. In the latter compartment, cargo proteins are separated from ER resident proteins and transported to the cis-Golgi along microtubules. Conversely, ER resident proteins and cargo receptors, that display ER-retrieval signals, are returned to the ER via COPI vesicles. Following cotranslational translocation, proteins destined to the secretory pathway acquire their native conformation within the ER. A robust quality control system operates in the ER to ensure that only properly folded proteins are allowed into transport vesicles . Nascent cargo is retained and/or not recognized by the export machinery until the cargo is fully folded and assembled [14, 15].
COPII complex assembly and evolution
Most of the sequential assembly of COPII machinery has been well defined in yeast. COPII recruitment is initiated by the activation of the small GTPase Sar1 by its ER-localized guanine exchange factor (GEF), Sec12. Sar1 initiates vesicle formation on ER membranes through the exchange of GDP for GTP, which induces tight membrane association of Sar1 and the subsequent recruitment of the heterodimeric complex comprising of Sec23/24 COPII components. Sec23 is a GTPase activating protein (GAP) that stimulates the enzymatic activity of Sar1, whereas Sec24 is the adaptor protein that captures specific cargo into the nascent vesicle. The Sar1-GTP/Sec23/Sec24 “pre-budding” complex in turn recruits the Sec13/Sec31 heterotetramer, which forms the outer layer of the COPII coat, a flexible coat cage that can accommodate various sizes of vesicles, and likely functions to cross-link adjacent prebudding complexes and complete the vesicle biogenesis process [16, 17] (Fig. 2). Two elegant studies [7, 18] have identified many of the molecular steps in the anterograde protein trafficking; however, they remained confined to unicellular yeast or mammalian cells in culture as model systems. Less is known about the behavior of the secretory pathway within the context of the entire multicellular organism and how its malfunction might influence development and organ homeostasis. The increase in size of eukaryotic cell was accompanied by an increase in complexity of the molecular machinery of these transport routes, i.e., number and types of coat proteins, regulatory GTPases, fusion and recruitment factors . One possible way in which evolution modifies function is to develop protein orthologues and isoforms, i.e., by gene duplication and/or by alternative mRNA splicing. Mammalian orthologues have been identified for each of the five core COPII components and, in some cases, multiple isoforms of these proteins exist, each encoded by a different gene. These are generally indicated by an alphabetical suffix. Two mammalian isoforms of Sar1 and Sec23, and four mammalian isoforms of Sec24 have been reported [20-23]. Only one form of mammalian Sec13 has been described . To date, only two mammalian proteins corresponding to the Sec31p yeast protein, Sec31A [25, 26] and Sec31B , have been identified.
COPII-related human genetic disorders
Presently, mutations in three components of the COPII core trafficking machinery have been assigned to human genetic disorders: they are the Chylomicron Retention Disease (CMRD), the Cranio-lenticulo-sutural dysplasia (CLSD), and the Congenital Dyserythropoietic Anemia type II (CDA II). We propose a classification of these diseases on the basis of the major clinical findings. However, this classification is only for a didactic purpose. So, we classified CMRD and CLSD as non-hematological disorders; conversely, CDA II will be discussed in the same section of the Combined Deficiency of Factor V and VIII (F5F8D), which is due to defects of F5/F8 selective cargo receptor (Table 1).
|Disease||Gene||Chromosome Localization||Protein function||Inheritance||Clinical phenotype||Prevalence|
|Cranio-lenticulo-sutural dysplasia (CLSD)||SEC23A||14q21.1||GAP||AR||Delay in closure of fontanels, sutural cataracts, facial dysmorphism, skeletal efects||Unknown|
|Congenital Dyserythropoietic Anemia type II (CDA II)||SEC23B||20p11.2||GAP||AR||Anemia, jaundice, low reticulocyte count, splenomegaly, hemochromatosis||0.7/100,0000a|
|Chylomicron Retention Disease (CMRD)||SAR1B||5q31.1||GTPase||AR||Malabsorption of fat, hypobetalipoproteinemia||Unknown|
|Combined Deficiency of Factor V and VIII (F5F8D)||LMAN1||18q21.3||Transmembrane lectin, Soluble protein||AR||Bleeding symptoms, epistaxis, menorrhagia, and excessive bleeding during or after trauma||1/100,000–1/100,00,00|
Chylomicron Retention Disease (CMRD) (OMIM 246700), or Anderson's disease, is a fat malabsorption disorder, in which enterocytes fail to secrete chylomicrons in lymph after a fat meal. This defect is usually diagnosed in infants presenting with failure to thrive, chronic diarrhea, low plasma vitamin E levels, hypocholesterolemia and hypobetalipoproteinemia with selective absence of apoB48 in the post prandial state . The low plasma lipid levels and low fat-soluble vitamin levels commonly cause mild peripheral neuropathy with diminished or absent deep tendon reflexes and definite or borderline mental retardation. However, neurological signs may develop more frequently later in untreated individuals [29, 30]. A typical histological finding in the intestinal biopsy has been noted, with a distinctive white stippling, resembling hoar frosting, covering the mucosal surface of the small intestine. The enterocytes contain accumulations of large lipid droplets free in the cytoplasm as well as membrane-bound lipoprotein-sized structures [30-32]. In 1987, in vitro studies of small intestinal explants from CMRD patients showed a normal apoB-48 protein synthesis, but impaired chylomicron synthesis in view of the altered glycosylation. The authors postulated a defect in the formation and secretion of chylomicrons resulting from a defect in glycosylation ; this observation was the prelude to the identification of the causative gene, 16 years later. Indeed, after the exclusion of APOB as causative gene , in 2003 Jones et al. demonstrated that the CMRD is caused by missense substitutions in SAR1B, one of two paralogous Sar1 proteins in humans  (Fig. 2). SAR1B mutations lead to an impaired chylomicron trafficking between ER and Golgi, with a subsequent accumulation of prechylomicron transport vesicles in the cytoplasm of the enterocytes . CMRD is a very rare recessively inherited disease with less than 50 cases having been reported in the literature. The diagnosis is often delayed because symptoms are nonspecific, but recently clinical guidelines for the diagnosis, follow-up, and treatment have been proposed .
The Boyadjiev-Jabs syndrome or Cranio-lenticulo-sutural dysplasia (CLSD) (OMIM 607812) is an autosomal recessive disorder characterized by late-closing fontanels, sutural cataracts, facial dysmorphisms, and skeletal defects. CLSD was originally mapped to chromosome 14q13-q21 in five males and one female from a large consanguineous Saudi Arabian family of Bedouin descent . Subsequently, it has been showed that this disorder arises from a missense substitution (F382L) in SEC23A, a gene encoding one of two paralogous proteins of the inner layer of COPII vesicle  (Fig. 2). Cell-free vesicle budding assays show that the F382L-SEC23A protein retains many aspects of wild-type function, including Sec24 binding, membrane binding and intrinsic GAP activity. However, the mutant protein shows reduced recruitment of the Sec13-Sec31 outer coat complex, especially when paired with SAR1B, indicating distinct affinities of the two human Sar1 paralogs for the SEC13-SEC31A complex .
Studies on Sec23a-deficient embryos of zebrafish suggested that disrupted ER export of the secretory proteins required for normal morphogenesis, such as collagen type II, accounts for CLSD . This alteration leads mutant chondrocytes to accumulate large amount of matrix proteins into extended ER compartments and proteasomes, unable to transport them to the Golgi for posttranslational modifications and eventually to the extracellular matrix. Lang et al. hypothesize that chondrocytes sense the absence of extracellular matrix maintaining abnormally high transcription of collagen genes. This excessive level of transcription might further exacerbate accumulation of unfolded proteins in the ER and trigger the response of the ER quality control system . Similarly to those observed in Sec23a-deficient zebrafish embryos, nonsense mutations in Sec23 were also found to have serious developmental consequences in worms .
Congenital dyserythropoietic anemia type II
Congenital Dyserythropoietic Anemia type II (CDA II) (OMIM 224100) is an autosomal recessive disorder characterized by ineffective erythropoiesis: this is suspected if there are symptoms and signs of increased hemoglobin turnover, such as mild jaundice due to indirect hyperbilirubinemia and low or absent haptoglobin, but reticulocytosis does not correspond to the degree of anemia. The main clinical findings of CDA II are moderate to severe normocytic or microcytic anemia, chronic or intermittent jaundice, splenomegaly . CDA II is associated with a well-defined morphological phenotype: peripheral blood smears show distinct anisopoikilocytosis with basophilic stippling and a few (occasionally binucleated) mature erythroblasts. Bone marrow shows 5–10 times more erythroblasts than normal (erythroid hyperplasia) [43, 44]. Early erythroblasts are relatively normal, but more than 10% of all erythroid cells are binucleated with equal size of two nuclei or multinucleated . In addition, upon electron microscopy, vesicles of ER appear to be running beneath the plasma membrane of CDA II erythroblastic cells . Similarly to others COPII-related human disorders [35, 37], CDA II shows a number of abnormalities affecting glycosylation and/or levels of erythrocyte glycoconjugates. The most useful for diagnosis is the hypoglycosylation of the erythrocyte anion exchanger 1 (AE1 or band 3), which represents a key for the diagnosis  and suggests a defect in vesicles trafficking. As already stated, it seems that the altered glycosylation lead the accumulation of large amount of unfolded proteins in the ER and triggers the response of the ER quality control system, the unfolded protein response (UPR) system . The reduced glycosylation is associated with a decreased activity of AE1 in red blood cells from CDA II patients; furthermore the CDA II erythrocytes were found to contain higher amounts of aggregate AE1 than control erythrocytes . Aggregated AE1 has been reported to bind naturally occurring antibodies, possibly mediating the phagocytic removal of red blood cells . These results suggested that the hemolysis found in CDA II patients may be ascribed to clustering of the AE1, leading to IgG binding and phagocytosis. All types of CDA share a high incidence of splenomegaly, cholelithiasis and iron overload , which in turn leads to secondary haemochromatosis: this represents the most important long-term complication encountered by patients after the first years of life. Of note, iron overload is not dependent on (albeit enhanced by) transfusions. Indeed, even in patients with mild or moderate anemia, ferritin levels should be checked at least annually, because iron overload may approach risk levels at any age. Haemochromatosis can lead to organ damage if not recognized and properly treated [50, 51]. Splenomegaly becomes apparent in 75% of all patients in the first 3 decades of life . The efficiency of splenectomy is still not established well; however, reduced hypoglycosylation of band 3 seems to have a relevant role in spleen removal of red blood cells. The main benefit of splenectomy is abrogation of transfusion requirements and increase of the hemoglobin concentration in severe cases .
The prevalence of CDAs in Europe has been recently assessed . After this evaluation, CDA II seems to be the most frequent form of CDAs. The geographic distribution of affected patients suggests a higher frequency of the gene in Italy and in the Mediterranean countries as compared with central and northern Europe. The combined prevalence of CDA I and CDA II (based on all cases reported in the last 42 years) varies widely among European regions, with minimal values of 0.04 cases/million in Scandinavia and the highest value in Italy (2.49/million). The regional distribution of the Italian patients demonstrated a clustering in Southern Italy . CDA II (367 cases) is relatively more frequent than CDA I (122 cases), with an overall ratio of ∼3.0.
The CDA II locus was originally mapped to 20q11 . However, in a refined contig build (build 36.3), the markers with the highest CDA II lod scores overlap the minimal homozygosity region on the short arm of chromosome 20 . With the assumption that the cis, median and trans N-glycan Golgi processing of erythroblast glycoproteins was impaired, the SEC23B gene became a likely candidate for CDA II [55, 56]. SEC23B encodes the second paralogous protein of the inner layer of COPII vesicle (Fig. 2). To date, 59 different causative mutations in SEC23B gene have been described, localized along the entire coding sequence of the gene [55-60]. The vast majority of patients had two mutations (in the homozygous or compound heterozygous state), in accordance with the pattern of autosomal recessive inheritance. Homozygosity for nonsense mutations in SEC23B gene must be lethal since no patient with such a genotype has ever been observed. The association of one missense mutations and one nonsense mutation tends to produce a more severe presentation than two missense mutations. Although most of the mutations in SEC23B gene are the results of sporadic and independent events, four mutations accounted for more than 50% of mutated alleles, which is a guide for a rational molecular diagnosis [57, 58]. A clusterization of these mutations seems to be relevant in Southern Italy, where a founder effect has been observed , accordingly to the regional distribution of the Italian patients.
If mutations in SEC23B gene explain the impaired processing of CDA II erythroblast glycoproteins, it is not clear how these impede the ultimate cell division along erythroid differentiation, generating binucleated erythroblasts. Interestingly, SEC23B was identified as a component of midbody, a transient “organelle-like” structure remnant of cell division just prior to abscission . It remains to be established, whether SEC23B plays an active role in erythrocyte midbody assembly or deconstruction or whether glycosylation impairment indirectly affects cytokinesis.
CDA II and CLSD are caused by mutations in the two human paralogs of the same COPII component. So, how SEC23A e SEC23B lead to specific phenotypes? In CLSD, primary calvarial osteoblasts showed a very low level of SEC23B, suggesting these cells are specifically affected because of insufficient SEC23B protein available to provide normal SEC23 function. Conversely, unaffected tissues in CLSD patients might retain normal function through expression of sufficient SEC23B to complement the loss of SEC23A function . Similarly, in CDA II, it was showed an increased SEC23B expression during in vitro erythroid differentiation, 5–7 fold over SEC23A expression, whereas no gross RNA expression difference between the paralogs in primary dermal fibroblasts was observed . However, studies on zebrafish morphants showed that both Sec23 genes carry specific but partially redundant roles in craniofacial cartilage maturation [40, 56]: in addition, sec23b morphants show a typical erythroid phenotype, with a significant increase in immature, binucleated erythrocytes .
Combined deficiency of factor V and VIII
An alternative mechanism to protein secretion COPII-mediated involves selective packaging of secreted proteins with the help of specific cargo receptors (Fig. 3). A good example of this is the resulting defects in secretion of blood clotting factors in patients with F5F8D (OMIM 227300). This is an autosomal, recessive bleeding disorder that is distinct from the co-inheritance of deficiency of both FV and FVIII. It is characterized by a mild-to-moderate bleeding tendency manifested during or after trauma, tooth extraction, surgery, labor, and abortions. Menorrhagia is common, but hematuria, gastrointestinal, and intramuscular bleeding are rare. Affected individuals are characterized by concomitantly low levels of both FV and FVIII, usually between 5 and 30% of normal values, with normal platelet count, prolonged prothrombin time (PT), and partial thromboplastin time tests (PTT) [62, 63]. Congenital F5F8D is estimated to be extremely rare, affecting males and females in equal numbers. In the general population, increased frequency is associated with consanguineous marriage. Indeed, the highest reported occurrence of F5F8D is among Middle Eastern Jews and non-Jewish Iranians (1:100,000), where customary consanguineous marriages are frequent . However, this disorder may be significantly under-diagnosed because of the often mild bleeding symptoms, or misdiagnosed as single factor deficiencies in many countries with limited hematology/genetics infrastructure .
The majority (70%) of F5F8D patients have mutations in LMAN1 , with the remaining subset of patients resulting from mutations in MCFD2 . To date, at least 51 mutations affecting the LMAN1 and MCFD2 proteins have been described, 70% of which are located in the LMAN1 gene . Almost all LMAN1 mutations reported to date are null mutations; in contrast, in the MCFD2 gene have been identified mostly missense mutations . Although patients with LMAN1 mutations and patients with MCFD2 mutations are considered clinically indistinguishable, a genotype-phenotype correlation became evident with the comparison of a large number of patients with known mutations in either of the genes. Indeed, patients with MCFD2 mutations tend to have FV and FVIII levels at a lower range than patients with LMAN1 mutations .
LMAN1 (also known as ERGIC-53) is a mannose-selective lectin recycling from the ER to the ERGIC. MCFD2 (multiple coagulation factor deficiency 2) is a small, soluble protein with two Ca2+-binding motifs. Both proteins form a stable Ca2+-dependent complex with 1:1 stoichiometry that specifically aids in the transport of glycosylated FV and FVIII from the ER to the Golgi compartment. The intracellular localization of this complex at the ERGIC suggests a specific cargo receptor function for FV and FVIII in the early secretory pathway [69, 70]. Cargo receptors are transmembrane proteins, required for the efficient ER to Golgi transport of many soluble secretory proteins. They also contain ER exit signals on the cytoplasmic side that are recognized by the SEC24 component of COPII . To date, most cargo receptors are identified in yeast . However, the LMAN1-MCFD2 protein complex is the only well-characterized cargo receptor in mammalian cells . The majority of LMAN1 is localized to the ERGIC at steady state, it also cycles between the ER and ERGIC in live cells. This is achieved through signal peptides, ER exit and ER retrieval motifs, located at the C-terminal of the protein that interact with COPII and COPI respectively. Conversely, MCFD2 lacks the ER exit and retrieval signals; thus, it requires LMAN1 binding for proper intracellular localization [66, 67].
Given the ubiquitous expression pattern of LMAN1 and MCFD2 and the presence of their orthologues in lower eukaryotes without a blood clotting system, additional cargo proteins that are targeted by this complex probably also exist. Accordingly, cathepsin C, cathepsin Z and 1-antitrypsin have also been reported as potential cargo proteins dependent on LMAN1 for efficient secretion [73, 74]. However, LMAN1-deficient mice, with reduced FV and FVIII plasma levels to ∼50% of wild-type mice, show no differences to wild type mice in the levels of cathepsin C and cathepsin Z in liver lysates or α1-antitrypsin levels in plasma . What are the unique features of FV and FVIII that enable them to both interact with the LMAN1-MCFD2 complex? FV and FVIII are two plasma glycoproteins, which share a functionally dispensable domain (B-domain) that is heavily glycosylated. B-domain deleted FVIII exhibits markedly reduced binding to the LMAN1-MCFD2 complex , suggesting an interaction between the lectin LMAN1 and sugar side chains of the heavily glycosylated B domains of FV and FVIII. However, unglycosylated FVIII can still interact with the LMAN1-MCFD2 complex , suggesting that protein–protein interactions are also involved .