André Verbert

Glycoproteins from insect cells: sialylated or not?

Abstract Our growing comprehension of the biological roles of glycan moieties has created a clear need for expression systems that can produce mammaliantype 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 Nglycan species found on these glycoproteins are (Galß1,3)GalNAcOSer/Thr and Man3(Fuc)GlcNAc2-NAsn, 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 glycoconjugates. While most work indicates that insect cellderived 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 glycobiologists 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 baculovirusinsect cell system will require metabolic engineering efforts to extend the native protein glycosylation pathways of insect cells.

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.

Multiplex RT-PCR method for the analysis of the expression of human sialyltransferases: application to breast cancer cells.

In many cases of human cancer, the appearance of hypersialylated glycan structures is related to a precise stage of the disease; this may depend on altered regulation of one or more sialyltransferases genes. Since several distinct sialyltransferase enzymes arising from different unique genes transfer sialic acid residues in the same linkage onto the same acceptor, it is impossible to precisely determine which enzyme is involved in the observed phenotype based on enzymatic assays. We have developed a very sensitive and highly reproducible multiplex reverse transcriptase-polymerase chain reaction technique in order to monitor the expression of four human sialyltransferases genes ST6Gal I, ST3Gal I, ST3Gal III and ST3Gal IV in small cell samples. Multiplex PCR amplification using specific primers for each sialyltransferase and detection of amplification products by polyacrylamide gel electrophoresis is a method that is fast and easy to handle and has proven to be useful for establishing sialyltransferase patterns of expression in breast immortalized cell line HBL100 as well as in breast cancer cell lines MCF-7/6, MCF-7/AZ and MDA.

New evidence for cell surface galactosyltransferase

Since Roseman [ 1 ] has proposed implications of ectoglycosyltransferase systems in intercellular adhesion and cell recognition, many works tend to demonstrate the occurrence of cell surface glycosyltransferase ([2,7] and for review, see ref. [8] ). Except for ectosialyltransferase for which ultrastructural evidence has been obtained [9] the biochemical demonstration for ectoglycosyltransferase is not without certain failure. Recently, Keenan and Morre [lo] reviewed the different reasons which can be misleading to conclusive demonstration for the occurrence of this enzymatic system on the outer surface of the plasma membrane. One way to characterize these enzymes is to isolate plasma membranes, however, this approach may be faulty by the contamination with intracellular membrane associated enzymes. Moreover it does not prove the external orientation of the active sites. These are the main reasons why most authors deal with intact cell systems. As it has been pointed out by Keenan and Morre [lo] , incubation of cells with nucleotide sugars with or without exogenous acceptors leads to the following causes of errors: (i) precursor hydrolysis and entry of the free carbohydrate into the cells, (ii) release or secretion of intracellular glycosyltransferases, (iii) exogenous acceptors glycosylation may be due to phagocytosis or pinocytosis and later excretion in the incubation medium. Being aware of these facts, we have developed a new methodology to demonstrate clearly the occurrence of ectogalactosyltransferase. The use of acceptors coupled on Sepharose beads far larger in size than the cells allows us to eliminate the third remark. Measurement of the galactosyl transferase activity in the post incubation supernatant medium allows us to answer the second point. Finally, separation of cells from the insoluble acceptors after incubation enables us to discriminate between the glycosylation of the acceptor and the incorporation by the cell.

Detection of ectosialyltransferase activity using whole cells. Correction of misleading results due to the release of intracellular CMP-N-acetylneuraminic acid

An inhibitory effect due to broken cells is observed when sialyltransferase (CMP-N-acetylneuraminate:D-galactosyl-glycoprotein N-acetylneuraminyltransferase, EC 2.4.99.1) is measured with mixture of intact and homogenized lymphocytes. This intracellular inhibitory factor ib purified and characterized as CMP-N-acetylneuraminic acid (CMP-NeuNAc) by its behavior in various chromatographic and electrophoretic systems and by its susceptibility to CMP-NeuNAc hydrolase. This endogenous CMP-NeuNAc leads to an isotopic dilution of the exogenous labelled CMP-NeuNAc explaining the apparently lower activity of homogenate when compared to whole cells. Consequently, the radioactivity bound to acceptors may not be related to a known number of sialyl residues transferred, calling into question the validity of comparing the incorporation of [14C]NeuNAc by homogenate and whole cells in order to assign sialyltransferase activity to ectoenzyme. A new approach is developed to detect ectoglycosyltransferases with whole cells, taking into account that both intracellular enzymes and endogenous precursor may be introduced by the small percentage of broken cells.

Effect of cyclic nucleotides in in vitro assays of rat liver galactosyltransferase

Experiments with rat liver microsomal galactosyltransferase has been developed to test the effect of cyclic nucleotides on the transfer activity. An overall stimulation is observed when cAMP or cGMP (concentration higher that 10(-6) M) are added to the incubation medium. However, more detailed experiments show that the cyclic nucleotides do not act as direct effectors of the enzyme but present the precursors degradation by the glycosylnucleotide pyrophosphatases.

5'-TriphosphaTe termini of RNAs made in vivo and in vitro by HeLa and KB cell DNA-dependent RNA polymerases.

Summary This work shows that the 5′-termini of HeLa and KB cell RNAs contain exclusively purine nucleoside triphosphates with a predominance of GTP. Experiments with actinomycin D, at a concentration which inhibits specifically rRNA synthesis, indicate an extensive decrease of GTP at 5′-ends which means that the major part of nucleolar RNAs, presumably including high molecular weight rRNA precursor, start with GTP. The relative maintenance of ATP at 5′-termini under actinomycin D treatment suggests that an important proportion of nucleoplasmic RNAs is initiated with ATP. These results are supported by further data obtained in vitro with KB cell RNA polymerases I and II.

Viral neuraminidase and cellular ectosialyltransferase in human lymphoblastoid cells infected with influenza virus

In human lymphoblastoid cells, infected with an influenza virus, Fowl Plague Virus (FPV), glycoproteins (such as secreted IgM) are hyposialylated, through the action of viral neuraminidase. In this study, the modulation of the cellular ectosialyltransferase activity during viral infection was investigated. This activity was detectable in FPV-infected cells, was shown to be 2.5-fold higher than that of uninfected cells, and to be able to restore, at least partially, the level of sialylation of the cell surface acceptors.

Inhibition of glycosyltransferases by bis-(p-nitrophenyl)phosphate: general effect and relation to their membrane integration.

The effect of bis-(p-nitrophenyl)phosphate on various glycosyltransferases involved in protein glycosylation (sialyl-, fucosyl-, galactosyl-, mannosyl- and glucosyltransferases) have been studied using crude enzyme preparations solubilized from rat spleen lymphocytes. Bis-(p-nitrophenyl)phosphate appears as a common inhibitor for every glycosyltransferase reaction utilizing sugar nucleotides as direct donors. In most cases 10 mM inhibitor is sufficient to obtain a 90 per cent inhibition. Kinetic studies achieved with a purified galactosyltransferase preparation reveal that bis-(p-nitrophenyl)phosphate exerts a competitive inhibition towards UDP-galactose binding. Concerning membrane-bound enzymes, the interaction of bis-(p-nitrophenyl)phosphate depends on its accessibility to the enzyme active site. This is shown by the different effect obtained with two UDP-Glc utilizing membrane-bound enzymes : UDP-Glc : phospho-dolichyl glucosyltransferase and UDP-Glc : ceramide glucosyltransferase : the first one not being affected but the second one being markedly inhibited under the same condition, although both are inhibited when the membrane environment is disturbed by detergent. Bis-(p-nitrophenyl)phosphate appears to be a tool to study membrane topology of glycosyltransferases.

Comparative Study of Ectogalactosyl- and Ectosialyl-transferases of Lymphocytes

Publisher Summary Ectoglycosyltransferases are defined as membrane-bound enzymes whose active sites are accessible from the outside of the cell. Ectoglycosyltransferase activities may be studied by the incubation of whole cells with labeled nucleotide–sugar, with or without exogenous acceptors. However, this method leads to several causes of errors. This chapter discusses different methodologies to clearly demonstrate the presence of ectogalactosyl- and ectosialyl-transferases at the outer surface of lymphocytes by (1) the use of acceptors coupled to Sepharose beads far larger in size than the cells, to eliminate phagocytosis, (2) the addition of nucleoside-5-mono-phosphate, of phloridzin, and of excess unlabeled sugar to minimize the entry of radioactive sugar into the cells and to allow its discrimination from the enzymic incorporation on the cell surface, and (c) the measurement of the glycosyltransferase activity in the post-incubâtion supernatant medium to check for an eventual release or secretion of intracellular enzyme. The use of these methodologies proves the occurrence of ectogalactosyl- and ectosialyl-transferase on the lymphocyte outer-surface.