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.

Our growing comprehension of the biological roles of glycan moieties has created a clear need for expression systems that can produce mammalian-type glycoproteins. In turn, this has intensified interest in understanding the protein glycosylation pathways of the heterologous hosts that are commonly used for recombinant glycoprotein expression. Among these, insect cells are the most widely used and, particularly in their role as hosts for baculovirus expression vectors, provide a powerful tool for biotechnology. Various studies of the glycosylation patterns of endogenous and recombinant glycoproteins produced by insect cells have revealed a large variety of O-and Nlinked glycan structures and have established that the major processed O-and N-glycan species found on these glycoproteins are (Galβ1,3)GalNAc-O-Ser/Thr and Man3(Fuc)GlcNAc2-N-Asn, respectively. However, the ability or inability of insect cells to synthesize and compartmentalize sialic acids and to produce sialylated glycans remains controversial. This is an important issue because terminal sialic acid residues play diverse biological roles in many glyco-conjugates. While most work indicates that insect cell-derived glycoproteins are not sialylated, some wellcontrolled studies suggest that sialylation can occur. In evaluating this work, it is important to recognize that oligosaccharide structural determination is tedious work, due to the infinite diversity of this class of compounds. Furthermore, there is no universal method of glycan analysis; rather, various strategies and techniques can be used, which provide gly-cobiologists with relatively more or less precise and reliable results. Therefore, it is important to consider the methodology used to assess glycan structures when evaluating these studies. The purpose of this review is to survey the studies that have contributed to our current view of glycoprotein sialylation in insect cell systems, according to the methods used. Possible reasons for the disagreement on this topic in the literature, which include the diverse origins of biological material and experimental artifacts, will be discussed. In the final analysis, it appears that if insect cells have the genetic potential to perform sialylation of glycoproteins, this is a highly specialized function that probably occurs rarely. Thus, the production of sialylated recombinant glycoproteins in the baculovirus-insect cell system will require metabolic engineering efforts to extend the native protein glycosylation pathways of insect cells.

We have analysed the mucins synthesized by the HT-29 MTX cell subpopulation, derived from the HT-29 human colon carcinoma cells through a selective pressure with methotrexate (Lesuffleur et al., 1990, Cancer Res 50: 6334-43), in the presence of benzyl-N-acetyl-alpha-galactosaminide (GalNAc alpha-O-benzyl), which is a potential competitive inhibitor of the beta 1,3-galactosyltransferase that synthesizes the T-antigen. The main observation was a 13-fold decrease in the sialic acid content of mucins after 24 h of exposure to 5 mM GalNAc alpha-O-benzyl. This effect was accompanied by an increased reactivity of these mucins to peanut lectin, testifying to the higher amount of T-antigen. The second observation was a decrease in the secretion of the mucins by GalNAc alpha-O-benzyl treated cells. The decrease in mucin sialylation was achieved through the in situ beta-galactosylation of GalNAc alpha-O-benzyl into Gal beta 1-3GalNAc alpha-O-benzyl, which acts as a competitive substrate of Gal beta 1-3GalNAc alpha 2,3-sialyltransferase, as shown by the intracellular accumulation of NeuAc alpha 2-3Gal beta 1-3GalNAc alpha-O-benzyl in treated cells.

This paper presents kinetic and structural analyses of oligosaccharide material released during glycosylation in permeabilized Chinese hamster ovary cells incubated with sugar nucleotides. Permeabilized cells released 30 times more oligosaccharide material than metabolically labelled cells, normalized to the amount of labelled glycoprotein acceptor, making this an amenable system for study. Fifteen to forty per cent of the oligosaccharide material released by permeabilized cells was oligosaccharide-phosphate, depending on the nature and amount of the oligosaccharide-lipids synthesized. The oligosaccharide-phosphates released were recovered in the cytosol, and were exclusively Man2Glc-NAc2P and Man5GlcNAc2P, released from oligosaccharide-lipids thought to be facing the cytosol. In contrast, the structures found as neutral oligosaccharide material were similar to those attached to newly synthesized glycoproteins, indicating that the oligosaccharides were subjected to the same processing enzymes whether or not they were protein bound. Importantly, the kinetics of the transfer to protein and the release of free neutral oligosaccharide were parallel, suggesting that the same enzyme was responsible for both processes. Structural analyses demonstrated that the same Man5GlcNAc2 structure was transferred to protein and released as free oligosaccharide. Neutral oligosaccharides were found in both the cytosol and the pellet; however, oligosaccharides with one GlcNAc residue at the reducing end (OS-Gn1) were found exclusively in the supernate. The major neutral oligosaccharide produced after 2 h of metabolic labelling was Man5GlcNAc and it was found in the cytosol.