The integrase of the human immunodeficiency virus type 1 (HIV-1) has been expressed in yeast in order to investigate its potential lethal effect mediated by DNA damage. To this end, we have constructed an expression plasmid containing the retroviral integrase gene under the control of the inducible promotor ADH2/GAPDH which is regulated by the glucose concentration of the medium. Haploid yeast strain W303-1A did not appear to be clearly sensitive to HIV-1 integrase expression. However, disruption of the RAD 52 gene, which is involved in the repair of double-strand DNA breaks, strongly increased the deleterious effects of the retroviral enzyme in this yeast strain. The diploid strain constructed with W303-1A and an isogenic strain of the opposite mating type also showed a strong sensitivity to the HIV-1 integrase. Under yeast culture conditions allowing moderate integrase synthesis, the deleterious effect was totally abolished by missense integrase mutations, which are known to abolish HIV-1 integrase activities in vitro. We conclude that the lethal phenotype due to HIV-1 integrase expression in yeast may be closely related to the HIV-1 integration reaction in infected human cells, and that yeast may be a useful tool to study the HIV-1 integration process and to screen drugs capable of inhibiting HIV-1 integration in vivo.

No abstract

N-glycosylation is important for the folding and quality control of membrane and secretory proteins. We used mutagenesis to introduce N-glycosylation sequons in recombinant proteins to improve their secretion in HEK293 cells. Seven recombinant proteins, with or without endogenous N-glycosylation sequons, were tested by this method. Our results indicate that N-glycosylation sequons located at the N-or C-terminal are glycosylated at high rates and thus the N-and C-terminal may be convenient sites for effectively attaching oligosaccharide chains. More importantly, introduction of oligosaccharide chains at such positions has been found to improve the secretion levels for the majority of the recombinant proteins in our studies, regardless of endogenous N-glycosylation, presumably by improving their folding in the endoplasmic reticulum. V V C 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 1468Prog., 25: -1475Prog., 25: , 2009 Keywords: N-glycosylation, protein expression, secretion, protein folding, mutagenesis, quality control IntroductionAsparagine glycosylation (N-glycosylation) is an important process for the delivery of secretory and membrane proteins to the extracellular space or cell surface. Thus, most secretory and membrane proteins contain asparagine-linked (N-linked) glycans, with one or more oligosaccharide chains attached to the polypeptide backbone. For some proteins, Nglycosylation is absolutely required for their secretion. For others, it may not be necessary. Nevertheless, oligosaccharide chains have been shown to be the important parts of the proteins and may play roles in the folding, transport, degradation, structure, stability, receptor-recognition, cellular localization, or other biological activities of the glycoproteins (reviewed in Refs. 1-3). Protein N-glycosylation takes place at the membrane of the endoplasmic reticulum (ER). The ER plays a primary quality control function to ensure the fidelity and proper folding of proteins before they travel out of the compartment, as incorrectly folded proteins can be toxic to the cell. A presynthesized oligosaccharide chain on a lipid carrier, dolichol, is transferred onto the asparagine residue in the NXS/T sequon of the nascent polypeptide by the oligosaccharyltransferase (OST) complex while translation is in process. 2 The attached oligosaccharide chains continue to be modified along the maturation pathway of the proteins in the ER and the N-glycosylation status will dictate the fate of the synthesized protein. The newly synthesized protein can proceed to secretion, aggregation, or degradation depending on the status and pattern of its N-glycosylation. [4][5][6] In the overexpression and production of recombinant proteins, the efficiency of N-glycosylation will affect the yield of the protein recovery and the circulation life of a pharmaceutical molecule. Although N-glycosylation is important, it is well known that not all potential glycosylation sites are attached with oligosaccharide chains. In our routine expression experi...

In a previous study (Xu, Z., Vo, L., and Macher, B. A. (1996) J. Biol. Chem. 271, 8818 -8823), a domain swapping approach demonstrated that a region of amino acids found in human ␣1,3/4-fucosyltransferase III (FucT III) conferred a significant increase in ␣1,4-FucT acceptor substrate specificity into ␣1,3-fucosyltransferase V (FucT V), which, under the same assay conditions, has extremely low ␣1,4-FucT acceptor substrate specificity. In the current study, site-directed mutagenesis was utilized to identify which of the eight amino acids, associated with ␣1,4-FucT acceptor substrate specificity, is/ are responsible for conferring this new property. The results demonstrate that increased ␣1,4-FucT activity with both disaccharide and glycolipid acceptors can be conferred on FucT V by modifying as few as two (Asn 86 to His and Thr 87 to Ile) of the eight amino acids originally swapped from FucT III into the FucT V sequence. Neither single amino acid mutant had increased ␣1,4-FucT activity relative to that of FucT V. Kinetic analyses of FucT V mutants demonstrated a reduced K m for Gal␤1,3GlcNAc (type 1) acceptor substrates compared with native FucT V. However, this was about 20-fold higher than that found for native FucT III, suggesting that other amino acids in FucT III must contribute to its overall binding site for type 1 substrates. These results demonstrate that amino acid residues near the amino terminus of the catalytic domain of FucT III contribute to its acceptor substrate specificity.␣1,3/4-Fucosyltransferases (FucTs) 1 can bind a wide variety of acceptor substrates (see Ref. 1 and references therein) and catalyze the synthesis of glycoconjugates with different immunological and functional properties including blood group antigens and selectin ligands (2-9). Interestingly, the acceptor substrate specificity of different forms of the human FucTs with highly homologous amino acid sequences can differ significantly (1, 10 -13). For example, FucT III has a very high level of activity with a type 1 disaccharide acceptor (Gal␤1, 3GlcNAc), whereas FucT VI appears to be a true ␣1,3-FucT and utilizes exclusively type 2 acceptors (1, 10 -15 substrate specificities when assayed in vitro with disaccharide acceptors despite the fact that they share Ͼ90% amino acid sequence homology (10 -13). In addition, some of these enzymes can bind the same acceptor substrates but produce different products. For example, mammalian cells transfected with FucT V can produce cell surface antigens recognized by anti-VIM 2 and anti-difucosyl-sLe x , whereas cells transfected with FucT VI express antigens recognized by antidifucosyl-sLe x but not anti-VIM 2 (11, 13). In a previous study, we demonstrated that truncated forms of FucT III and FucT V, containing ϳ300 amino acids and differing at less than 25 positions, have dramatically different acceptor substrate specificities when assayed with simple disaccharide substrates (16). Furthermore, we found that the swapping of an NH 2 -terminal segment of FucT III, containing eight amino acids u...

. Among the conserved residues in FucTs (including human, mouse, chicken, and zebra fish) are four cysteine (Cys) residues (Fig. 1). Two of these Cys residues are located near the N terminus and two near the C terminus of the catalytic domain. The Cys residues found at the C terminus also are conserved in FucTs from Caenorhabditis elegans (6). Among the human FucTs, FucTs III, V, and VI share substantial sequence homology. Within the catalytic domain only about 20 out of 300 amino acids vary among the three proteins. In addition, domain swapping experiments by our group (7) and Lowe and co-workers (9) have demonstrated that chimeric proteins composed of partial sequence from each of these FucTs are active, indicating that the minor differences in their amino acid sequences does not result in major alterations in their overall structure. Therefore, we have used two (FucT III and V) of these highly homologous proteins in the present study to evaluate the structure and functional significance of the highly conserved Cys residues.Protein chemistry experiments, coupled with mass spectrometry analyses, have been used to locate all peptides containing Cys residues in human FucT III, allowing them to be assigned either as being involved in a disulfide bond or as a free Cys residue, and identifying which Cys residues are bound to each other in disulfide linkages. The results support our (7) previously stated hypothesis that amino acids affecting acceptor substrate specificity of human FucT III and V, located near the N and C termini of the catalytic domain, are brought close together in space by disulfide bonds between these highly conserved Cys residues.To investigate the importance of these Cys residues, we have mutated each of these Cys residues in human FucT V and evaluated the activity and other properties of each resulting protein construct. The results demonstrate that these residues affect enzyme activity, but not the interaction of the protein with GDP-Fuc in three of the four cases. In the case of one of the mutant constructs (Cys 104 ) protein folding/stability is altered compared with the wild type protein.The amino acid sequences of FucTs have been predicted on the basis of cDNA sequences, but none of the amino acid se-