[Hereditary multiple exostosis complicated by spontaneous haemothorax]. / Hémothorax spontané compliquant une maladie des exostoses multiples.

Spontaneous haemothorax is a rare event in the general population. However some diseases, including hereditary multiple exostosis, have an increased incidence of this complication. We report the case of a 22 year old patient with hereditary multiple exostosis who presented with a right sided haemothorax due to an exostosis of the seventh rib. The pathophysiology of spontaneous haemothorax in the setting of hereditary multiple exostosis is discussed together with surgical treatment and long term follow up.

'Glyco-deglyco' processes during the synthesis of N-glycoproteins.

For the past 15 years, it has appeared increasingly evident that the N-glycosylation process was accompanied by the release of oligomannoside type oligosaccharides. This material is constituted of oligosaccharide-phosphates and of neutral oligosaccharides possessing one GlcNAc (OS-Gn1) or two GlcNAc (OS-Gn2) at the reducing end. It has been demonstrated that oligosaccharide-phosphates originated from the cleavage by a specific pyrophosphatase, of non-glycosylated cytosolic faced oligosaccharide-PP-Dol and chiefly the Man5GlcNAc2-PP-Dol. The Man5GlcNAc2-P, as the main product, is recovered in the cytosolic compartment and is further degraded to Man5GlcNAc1 by as for yet not depicted enzymes. In contrast, OS-Gn2 produced from hydrolysis of oligosaccharide-PP-Dol (presumably as a transfer reaction onto water) when the amount of protein acceptor is limiting, are generated into the lumen of rough endoplasmic reticulum (ER). They are further submitted to processing alpha-glucosidases and rough ER mannosidase and are (mainly as Man8GlcNAc2) exported into the cytosolic compartment. This material is further degraded into a single component, the Man5GlcNAc1: Man alpha 1-2Man alpha 1-2Man alpha 1-3 (Man alpha 1-6)Man beta 1-4GlcNAc by the sequential action of a cytosolic neutral chitobiase followed by cytosolic mannosidase. Furthermore, OS-Gn1 could have a dual origin: on the one hand, they originate from OS-Gn2 by the cytosolic degradation pathway indicated above; on the other hand, we will discuss a possible origin from the degradation or remodeling of newly synthesized glycoproteins. Considered first as a minor phenomenon, these observations have lead to the concept of intracellular oligomannoside trafficking, a process which results from more fundamental phenomena such as the control of the dolichol cycle, and the so-called quality-control of glycoprotein. In this review, we would like to describe the evolution of ideas on the origin, intracellular trafficking and putative roles of these oligomannosides released during the N-glycosylation process. We propose that these early stage 'glyco-deglyco' processes represent a way of control of N-glycosylation and of the fate of N-glycoproteins.

Retention and degradation of N-glycoproteins in the rough endoplasmic reticulum.

Recent studies have shown that newly synthesized proteins and glycoproteins are submitted to a quality control mechanism in the rough endoplasmic reticulum (ER). In this report we present two models: One model will illustrate a transient retention in rough ER leading to a further degradation of glycoproteins in the cytosol, (soluble alkaline phosphatase expressed in Man-P-Dol deficient CHO cells lines). The second model will illustrate a strict retention of glycoproteins in rough ER without degradation nor recycling through the Golgi (E1, E2 glycoproteins of Hepatitis C virus in stably transfected UHCV-11.4 cells and in infected Hep G2 cells). In both cases, oligomannoside structures are markers of these phenomena, either as free soluble released oligomannosides in the case of degradation, or as N-linked oligomannosides for strict retention in rough ER.

[Leiomyosarcoma and stromal tumor of the pancreas]. / Léiomyosarcome et tumeur stromale du pancréas.

A tumor of the head of pancreas was an incidental finding on US of two patients aged 52 and 61 years presenting with abdominal pain. The tumor was studied by computed tomography, MRI and endoscopic ultrasonography. Surgical biopsy of a liver lesion was performed in one case and partial duodenopancreatectomy was performed in the other case. Histological evaluation, including immunohistochemistry, showed leiomyosarcoma and stromal tumor respectively. Whatever the site of origin, most mesenchymal pancreatic tumors are hypervascular, heterogeneous and have a necrotic center.

[Contribution of spiral CT for the early diagnosis of hepato-cellular carcinoma in cirrhotic patients]. / Intérêt du scanner hélicoïdal dans le diagnostic précoce des carcinomes hépatocellulaires sur cirrhose.

PURPOSE: To evaluate the specificity of spiral CT for early diagnosis of small hepato-cellular carcinoma. PATIENTS AND METHODS: One or several hepatic hypervascular nodules, with a diameter smaller than or equal to 3cm and suspected of corresponding to small hepato-cellular carcinoma, were detected during the arterial phase of spiral CT in 55 patients. The diagnosis of the main nodule could be established in 31 patients by pathologic examination, elevated alpha-foeto-protein level or follow-up examination. RESULTS: Among the 31 studied nodules, 19 were hepato-cellular carcinomas (61%) and 12 were benign (39%). Seven (78%) of the 9 nodules>20mm and 12 (54%) of the 22 nodules<=20mmm were hepato-cellular carcinomas. Three benign lesions (2 angiomas and 1 pseudo-nodule of hemodynamic origin) were showing typical radiological characteristics which retrospectively allowed their identification. On the other hand, the appearance of the 9 other benign nodules (29%) was exactly the same as the one of the hepato-cellular carcinoma. CONCLUSION: Although, in cirrhotic patients, hepato-cellular carcinoma is the most frequent cause of detected nodules during the arterial phase of the spiral CT, 29% of hypervascular lesions<=3cm were benign in this study. In some cases, only follow-up examinations are able to confirm the diagnosis.

Truncated N-glycans affect protein folding in the ER of CHO-derived mutant cell lines without preventing calnexin binding.

The involvement of N-glycans in the folding of influenza virus hemagglutinin (HA) was analyzed in two CHO-derived glycosylation mutants exhibiting a thermosensitive defect for secretion of human placental alkaline phosphatase. Truncated Man(5)GlcNAc(2)oligosaccharides with one or three glucose residues are attached to proteins of the MadIA214 and B3F7AP2-1 mutant cells, respectively. Newly synthesized proteins retained in these cells carry a Man(4)trimmed glycan generated by a mannosidase different from the ER mannosidases I and II and suggesting a recycling through the Golgi complex. The glucosidase inhibitor castanospermine affects the binding of HA folding intermediates to the lectin-like chaperone calnexin in B3F7AP2-1 but not in MadIA214 cells. We demonstrated that calnexin interacts in vivo with truncated Man(5)derivatives. In MadIA214 cells, this is only possible when Man(5)GlcNAc(2)on protein becomes reglucosylated. The pattern of intermediates seen during the folding of HA in the MadIA214 and B3F7AP2-1 mutant cell lines is different than in control cells. We also observed a variable occupancy of the seven glycosylation-sites. However, even under conditions that restore glycosylation of all sites, the folding intermediates of HA in the mutant cells still remain heterogeneous. Our results demonstrate that addition of truncated N-glycans interferes extensively with the folding of newly synthesized proteins in vivo.

Reciprocal relationship between alpha1,2 mannosidase processing and reglucosylation in the rough endoplasmic reticulum of Man-P-Dol deficient cells.

The study of the glycosylation pathway of a mannosylphosphoryldolichol-deficient CHO mutant cell line (B3F7) reveals that truncated Glc(0-3)Man5GlcNAc2 oligosaccharides are transferred onto nascent proteins. Pulse-chase experiments indicate that these newly synthesized glycoproteins are retained in intracellular compartments and converted to Man4GlcNAc2 species. In this paper, we demonstrate that the alpha1,2 mannosidase, which is involved in the processing of Man5GlcNAc2 into Man4GlcNAc2, is located in the rough endoplasmic reticulum. The enzyme was shown to be inhibited by kifunensine and deoxymannojirimycin, indicating that it is a class I mannosidase. In addition, Man4GlcNAc2 species were produced at the expense of Glc1Man5GlcNAc2 species. Thus, the trimming of Man5GlcNAc2 to Man4GlcNAc2, which is catalyzed by this mannosidase, could be involved in the control of the glucose-dependent folding pathway.

Monoglucosylated oligomannosides are released during the degradation process of newly synthesized glycoproteins.

The Chinese hamster ovary mutant MI8-5 is known to synthesize Man(9)GlcNAc(2)-P-P-dolichol rather than the fully glucosylated lipid intermediate Glc(3)Man(9)GlcNAc(2)-P-P-dolichol. This nonglucosylated oligosaccharide lipid precursor is used as donor for N-glycosylation. In this paper we demonstrate that a significant part of the glycans bound to the newly synthesized glycoproteins in MI8-5 cells are monoglucosylated. The presence of monoglucosylated glycans on glycoproteins determines their binding to calnexin as part of the quality control machinery. Furthermore, we point out the presence of Glc(1)Man(5)GlcNAc(1) in the cytosol of MI8-5 cells. This indicates that part of the monoglucosylated glycoproteins can be directed toward a deglycosylation process that occurs in the cytosol. Besides studies on glycoprotein degradation based on the disappearance of protein moieties, MI8-5 cells can be used as a tool to elucidate the various step leading to glycoprotein degradation by studying the fate of the glycan moieties.

Hepatitis C virus glycoprotein complex localization in the endoplasmic reticulum involves a determinant for retention and not retrieval.

The hepatitis C virus (HCV) genome encodes two envelope glycoproteins (E1 and E2). These glycoproteins interact to form a noncovalent heterodimeric complex which in the cell accumulates in endoplasmic reticulum (ER)-like structures. The transmembrane domain of E2, at least, is involved in HCV glycoprotein complex localization in this compartment. In principle, ER localization of a protein can be the consequence of actual retention in this organelle or of retrieval from the Golgi. To determine which of these two mechanisms is responsible for HCV glycoprotein complex accumulation in the ER, the precise localization of these proteins was studied by immunofluorescence, and the processing of their glycans was analyzed. Immunolocalization of HCV glycoproteins after nocodazole treatment suggested an ER retention. In addition, HCV glycoprotein glycans were not modified by Golgi enzymes, indicating that the ER localization of these proteins is not because of their retrieval from the cis Golgi. Retention of HCV glycoprotein complexes in the ER without retrieval suggests that this compartment plays an important role for the acquisition of the envelope of HCV particles. A true retention in the ER was also observed for E2 expressed in the absence of E1 or for a chimeric protein containing the ectodomain of CD4 in fusion with the transmembrane domain of E2. These data indicate that, in HCV glycoprotein complex, the transmembrane domain of E2, at least, is responsible for true retention in the ER, without recycling through the Golgi.

Cytosolic deglycosylation process of newly synthesized glycoproteins generates oligomannosides possessing one GlcNAc residue at the reducing end.

Recent studies on the mechanism of degradation of newly synthesized glycoproteins suggest the involvement of a retrotranslocation of the glycoprotein from the lumen of the rough endoplasmic reticulum into the cytosol, where a deglycosylation process takes place. In the studies reported here, we used a glycosylation mutant of Chinese hamster ovary cells that does not synthesize mannosylphosphoryldolichol and has an increased level of soluble oligomannosides originating from glycoprotein degradation. In the presence of anisomycin, an inhibitor of protein synthesis, we observed an accumulation of glucosylated oligosaccharide-lipid donors (Glc3Man5GlcNAc2-PP-Dol), which are the precursors of the soluble neutral oligosaccharide material. Inhibition of rough endoplasmic reticulum glucosidase(s) by castanospermine led to the formation of Glc3Man5GlcNAc2(OSGn2) (in which OSGn2 is an oligomannoside possessing two GlcNAc residues at its reducing end), which was then retained in the lumen of intracellular vesicles. Thus they were protected during an 8 h chase period from the action of cytosolic chitobiase, which is responsible for the conversion of OSGn2 to oligomannosides possessing one GlcNAc residue at the reducing end (OSGn1). In contrast, when protein synthesis was maintained in the presence of castanospermine, glucosylated oligomannosides (Glc1-3Man5GlcNAc1) were recovered in cytosol. Except for monoglucosylated Man5 species, which are potential substrates for luminal calnexin and calreticulin, the pattern of oligomannosides was similar to that observed on glycoproteins. The occurrence in the cytosol of glucosylated species with one GlcNAc residue at the reducing end implies that the deglycosylation process that generates glucosylated OSGn1 from glycoproteins occurs in the cytosol.

Glycosylation of the hepatitis C virus envelope protein E1 is dependent on the presence of a downstream sequence on the viral polyprotein.

The addition of N-linked oligosaccharides to Asn-X-(Ser/Thr) sites is catalyzed by the oligosaccharyltransferase, an enzyme closely associated with the translocon and generally thought to have access only to nascent chains as they emerge from the ribosome. However, the presence of the sequon does not automatically ensure core glycosylation because many proteins contain sequons that remain either nonglycosylated or glycosylated to a variable extent. In this study, hepatitis C virus (HCV) envelope protein E1 was used as a model to study the efficiency of N-glycosylation. HCV envelope proteins, E1 and E2, were released from a polyprotein precursor after cleavage by host signal peptidase(s). When expressed alone, E1 was not efficiently glycosylated. However, E1 glycosylation was improved when expressed as a polyprotein including full-length or truncated forms of E2. These data indicate that glycosylation of E1 is dependent on the presence of polypeptide sequences located downstream of E1 on HCV polyprotein.

The transmembrane domain of hepatitis C virus glycoprotein E1 is a signal for static retention in the endoplasmic reticulum.

Hepatitis C virus (HCV) glycoproteins E1 and E2 assemble to form a noncovalent heterodimer which, in the cell, accumulates in the endoplasmic reticulum (ER). Contrary to what is observed for proteins with a KDEL or a KKXX ER-targeting signal, the ER localization of the HCV glycoprotein complex is due to a static retention in this compartment rather than to its retrieval from the cis-Golgi region. A static retention in the ER is also observed when E2 is expressed in the absence of E1 or for a chimeric protein containing the ectodomain of CD4 in fusion with the transmembrane domain (TMD) of E2. Although they do not exclude the presence of an intracellular localization signal in E1, these data do suggest that the TMD of E2 is an ER retention signal for HCV glycoprotein complex. In this study chimeric proteins containing the ectodomain of CD4 or CD8 fused to the C-terminal hydrophobic sequence of E1 were shown to be localized in the ER, indicating that the TMD of E1 is also a signal for ER localization. In addition, these chimeric proteins were not processed by Golgi enzymes, indicating that the TMD of E1 is responsible for true retention in the ER, without recycling through the Golgi apparatus. Together, these data suggest that at least two signals (TMDs of E1 and E2) are involved in ER retention of the HCV glycoprotein complex.

The transmembrane domains of hepatitis C virus envelope glycoproteins E1 and E2 play a major role in heterodimerization.

Oligomerization of viral envelope proteins is essential to control virus assembly and fusion. The transmembrane domains (TMDs) of hepatitis C virus envelope glycoproteins E1 and E2 have been shown to play multiple functions during the biogenesis of E1E2 heterodimer. This makes them very unique among known transmembrane sequences. In this report, we used alanine scanning insertion mutagenesis in the TMDs of E1 and E2 to examine their role in the assembly of E1E2 heterodimer. Alanine insertion within the center of the TMDs of E1 or E2 or in the N-terminal part of the TMD of E1 dramatically reduced heterodimerization, demonstrating the essential role played by these domains in the assembly of hepatitis C virus envelope glycoproteins. To better understand the alanine scanning data obtained for the TMD of E1 which contains GXXXG motifs, we analyzed by circular dichroism and nuclear magnetic resonance the three-dimensional structure of the E1-(350-370) peptide encompassing the N-terminal sequence of the TMD of E1 involved in heterodimerization. Alanine scanning results and the three-dimensional molecular model we obtained provide the first framework for a molecular level understanding of the mechanism of hepatitis C virus envelope glycoprotein heterodimerization.