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A partial length cDNA encoding farnesyl pyrophosphate synthetase (hpt807) has been isolated from a human fetal liver cDNA library in lambda gt11. DNA sequence analysis reveals hpt807 is 1115 bp in length and contains an open reading frame coding for 346 amino acids before reaching a stop codon, a polyadenylation addition sequence, and the first 14 residues of a poly(A+) tail. Considerable nucleotide and deduced amino acid sequence homology is observed between hpt807 and previously isolated rat liver cDNAs for farnesyl pyrophosphate synthetase. Comparison with rat cDNAs suggests that hpt807 is about 20 bp short of encoding the initiator methionine of farnesyl pyrophosphate synthetase. The human cDNA was cloned into a prokaryotic expression vector and Escherichia coli strain DH5 alpha F'IQ was transformed. Clones were isolated that express an active fusion protein which can be readily observed on protein gels and specifically stained on immunoblots with an antibody raised against purified chicken farnesyl pyrophosphate phosphate synthetase. These data confirm the identity of hpt807 as encoding farnesyl pyrophosphate synthetase. Slot blot analyses of RNA isolated from Hep G2 cells show that the expression of farnesyl pyrophosphate synthetase mRNA is regulated. Lovastatin increases mRNA levels for farnesyl pyrophosphate synthetase 2.5-fold while mevalonic acid, low-density lipoprotein, and 25-hydroxycholesterol decrease mRNA levels to 40-50% of control values.

We previously reported the isolation of a partial-length human fetal-liver cDNA encoding farnesyl diphosphate (FPP) synthase (EC 2.5.1.10) and the expression of an active FPP synthase fusion protein in Escherichia coli. The expressed human FPP synthase fusion protein has now been purified to apparent homogeneity by using two chromatographic steps. The purification scheme allowed the preparation of 1.8 mg of homogeneous protein from 149 mg of crude extract in a 64% yield with a 52-fold enrichment. A single band with a subunit molecular mass of 39 kDa was observed by Coomassie Blue staining after SDS/PAGE. A molecular mass of 78-80 kDa was calculated for the native form of the fusion protein by h.p.l.c. on a SEC-250 column, suggesting that the active fusion protein is a dimer. The purified fusion protein has FPP synthase condensation activities in the presence of both substrates, isopentenyl diphosphate and geranyl diphosphate. Enzyme activity was inhibited by a bisubstrate analogue of isopentenyl diphosphate and dimethylallyl diphosphate, and a small amount of higher prenyltransferase was observed. Michaelis constants for isopentenyl diphosphate and geranyl diphosphate were 0.55 and 0.43 microM respectively, and Vmax for synthesis of farnesyl diphosphate from these substrates was 1.08 mumol/min per mg. These results suggest that the structure and catalytic properties of the expressed FPP synthase fusion protein are virtually identical with those of the native human liver enzyme.

In To date, this type of sugar chain has not been observed in chicken ovalbumin. These differences in fine structure, between the oligosaccharides derived from ovalbumin secreted by L cells and those known to be present in the chicken egg glycoprotein, suggest that the cell type also plays a role in oligosaccharide processing.Based on extensive studies, the enzymatic steps involved in the biosynthesis of oligosaccharide chains of asparaginelinked glycoproteins have been elucidated (1). Initially, the oligosaccharide chains are synthesized on a lipid-linked carrier; subsequently, they are cotranslationally transferred as a unit to the appropriate asparagine residues of the nascent polypeptide chain. The asparagine to be glycosylated must be part of a specific tripeptide acceptor sequence, Asn-XaaSer/Thr, this sequence being necessary but not sufficient for glycosylation. After transfer to protein, the oligosaccharide chain is processed, yielding either simple high-mannose chains or, after more extensive processing, complex-type sugar chains. In a few instances, as in the case of ovalbumin, hybrid structures are found containing features common to both high-mannose and complex oligosaccharides. Although the basic steps of the glycosylation pathway for asparagine-linked glycoproteins are known, the factors governing glycosylation and processing remain ambiguous. Because we are interested in determining the influence of the protein structure and cell type on proper-site glycosylation and oligosaccharide chain processing, we have begun to study the expression of cloned glycoproteins in heterologous cells. In this report, we examine the glycosylation of a model glycoprotein, chicken ovalbumin, in a mouse L-cell/herpes simplex virus type 1 (HSV-1) system (2).Mature chicken ovalbumin has two potential glycosylation sites, Asn-293 and Asn-312, of which only the first site is glycosylated in vivo. Chicken ovalbumin is heterogeneous with respect to its oligosaccharide chains, and at least nine basic structures have been characterized (3-6). The oligosaccharides fall into two major categories, high-mannose and hybrid. Although the exact distribution of high-mannose and hybrid chains is uncertain, the sum total of hybrid oligosaccharide chains is believed to be approximately equal to that of the high-mannose oligosaccharides (6). As (2) have shown that these cells synthesize an ovalbumin precursor that is processed and secreted into the extracellular medium after superinfection of the cells with HSV-1. These cells contain a plasmid (pRB353) with ovalbumin genomic DNA located downstream from and in the same transcriptional orientation as the a-regulated promoter of a protein 4 from HSV-1.Cells were maintained at 34°C in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum and hypoxanthine (0.11 mM), aminopterin (0.45 ,uM), and thymidine (20.6 ,M). Radiolabeled ovalbumin was prepared essentially as described (2). HSV-1 ts502A305 carries a temperature-sensitive mutation such th...

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