A full-length cDNA, which encodes a human placental fructose-6-phosphate,2-kinase/ fructose-2,6-bisphosphatase, was constructed and expressed in Escherichia coli. The expressed protein, purified to homogeneity, showed a molecular weight of 58,000 by gel electrophoresis under denaturing conditions, compared to the deduced molecular weight of 59,410. The N-terminal sequence of 15 amino acids coincided with that of the deduced sequence. The active enzyme was a dimer as judged by molecular sieve filtration. The expressed enzyme was bifunctional with Vmax values of 142 and 0.2 milliunits/mg for the kinase and phosphatase activities, respectively. The phosphatase activity was extremely low, because one phosphatase active site residue was mutated, and consequently the kinase/phosphatase ratio was the highest among the known isozymes. Furthermore, the enzyme was phosphorylated by cAMP-dependent protein kinase, protein kinase C and also by [2-32P]fructose-2,6-bisphosphate. Phosphorylation by cAMP-dependent protein kinase and protein kinase C increased the maximal Fru-6-P,2-kinase activities by 1.8- and 1.1-fold, respectively. These results suggested that placental fructose-6-phosphate,2-kinase/ fructose-2,6-bisphosphatase is important in maintaining and regulating a relatively high rate of glycolysis in placenta.

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The cytotoxic T lymphocyte (CTL) response has recently been shown to play a role in protection against human immunodeficiency virus (HIV) and it is therefore thought that a vaccine against HIV must be able to elicit a CTL response. The development of a safe, effective adjuvant is very important because alum, the only adjuvant available for use in humans at present, can barely induce a response of this type. We demonstrate here that liposomes that contain an immunodominant peptide (15 amino acids) of the envelope glycoprotein gp120 of HIV-1 and that are coated with mannopentaose-dipalmitoylphosphatidylethanolamine conjugate induce a major histocompatibility complex class I-restricted CD8 + CTL response in mice with a single subcutaneous immunization, whereas non-coated liposomes do not. Since no damage to the skin at the injection site was caused by the liposomes, and since the oligomannose-coated liposomes consist of innocuous materials ubiquitously distributed throughout the human body, they may be highly suitable for use as a safe adjuvant in vaccines inducing a CTL response against HIV.z 1998 Federation of European Biochemical Societies.

Two independent cDNA clones encoding fructose 6-phosphate, 2-kinase/fructose 2,6-bisphosphatase were isolated from a human placental cDNA library. The deduced amino acid sequences showed that one of the clones, 2K-1, was almost identical to the rat testis isozyme and the other, 2K-3, was different from any known isozymes expressed in mammalian tissues. The results of Southern blot analysis suggested that clones 2K-1 and 2K-3 were encoded as single copy genes and located in different parts of the genome. Since open reading frames of the cDNA clones were not complete, we obtained the 5'-end of the clone 2K-3 cDNA using the 5'-rapid amplification of cDNA end method. The entire cDNA (HP; 1,756 bp) had a coding capacity of 519 amino acids (M(r) = 59,410), and putative phosphorylation sites for protein kinases A and C on the C terminus. Northern blot analysis using a fragment of the HP as a probe showed that a major band of 5.4 kb, significantly different in size from known isozyme mRNAs such as liver (2.1 kb), muscle (1.9 kb), heart (4.0 kb), and testis (2.0 kb), was present in poly(A)+RNA preparations of human first trimester and term placentae. These results strongly suggested that this 5.4 kb mRNA codes a novel isozyme of fructose 6-phosphate,2-kinase/fructose 2,6-bisphosphatase.

The placenta-type isozyme of human 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (HP2K, identical to PFKFB3) is expressed in a variety of cells and tissues such as placenta, brain, testis, liver, kidney, skeletal muscle, primary blood mononuclear cells and cancer cells. We observed previously that the enhancer region of the HP2K gene, which has been identified in the 5'-flanking region between -1265 and -1329, could respond to serum stimulation following the transfection of human choriocarcinoma BeWo cells with HP2K promoter-luciferase constructs. The HP2K enhancer region also contains two copies of the hypoxia-inducible factor-1 (HIF-1) binding motif (5'-ACGTG-3'). In this study we performed characterization of the HP2K gene expression in response to hypoxic conditions. Both electrophoretic mobility shift and co-transfection assays of the HP2K promoter-luciferase reporter with HIF-1 expression vectors indicated that HIF-1 binds to the hypoxia-responsive element (HRE) of HP2K, thereby upregulating its gene expression. In addition, we demonstrated using site-directed mutagenesis that a complete tandem repeat of the HIF-1 binding motif with a 4-bp interruption is required for full induction of HP2K expression (up to 22-fold) under hypoxic conditions, and that this response is much stronger than that of the erythropoietin (EPO) gene. These results suggest that the sequence 5'-ACGTGNNNNACGTG-3' in the HP2K enhancer is the authentic HRE consensus motif that mediates increased transcription, under hypoxic conditions, via HIF-1.