Most cancer cells rely on glycolysis to generate ATP, even when oxygen is available. However, merely inhibiting the glycolysis is insufficient for the eradication of cancer cells. One main reason for this is that cancer cells have the potential to adapt their metabolism to their environmental conditions. In this study, we investigated how cancer cells modify their intracellular metabolism when glycolysis is suppressed, using PANC-1 pancreatic cancer cells and two other solid tumor cell lines, A549 and HeLa. Our study revealed that glycolytically suppressed cells upregulated mitochondrial function and relied on oxidative phosphorylation (OXPHOS) to obtain the ATP necessary for their survival. Dynamic changes in intracellular metabolic profiles were also observed, reflected by the reduced levels of TCA cycle intermediates and elevated levels of most amino acids. Glutamine and glutamate were important for this metabolic reprogramming, as these were largely consumed by influx into the TCA cycle when the glycolytic pathway was suppressed. During the reprogramming process, activated autophagy was involved in modulating mitochondrial function. We conclude that upon glycolytic suppression in multiple types of tumor cells, intracellular energy metabolism is reprogrammed toward mitochondrial OXPHOS in an autophagy-dependent manner to ensure cellular survival.

The sperm receptor activity of pig zona pellucida has been previously shown to exist in one of the components, pig zona protein 3n (PZP3n), that can be purified after the removal of sialylated and/or sulfated N-acetylpoly(1actosamine) by digestion with endo-P-galactosidase. In this study, we examined whether N-linked or 0-linked carbohydrate chains are involved in the sperm receptor activity of pig zona pellucida. The elimination of N-linked carbohydrate chains from endo-a-galactosidase-digested PZP3a by digestion with N-glycanase markedly reduced its inhibitory effect on sperm-egg binding in an in vitro competition assay, whereas the elimination of 0-linked carbohydrate chains by alkali treatment hardly reduced the inhibitory effect. These results indicate that N-linked carbohydrate chains of PZP3a play a major role in mediating the sperm binding of zona pellucida in pig.Keywords: zona pellucida; N-linked carbohydrate chains; N-glycanase; sperm-egg binding.The zona pellucida, which surrounds mammalian oocytes, consists of 3-4 glycoproteins and plays an important role in species-specific gamete recognition and the blocking of polyspermy after fertilization [I, 21. Pig zona pellucida consists of three glycoproteins: pig zona protein 1 (PZPI ; 90-kDa glycoprotein composed of disulfide-bonded 65-kDa and 25-kDa polypeptides 13, 41); pig zona protein 3n (PZP3a; 55 kDa); pig zona protein 3P (PZP3p; 55 kDa). PZP3a and PZP3p copurify in the pig zona protein 3 (PZP3) fraction during gel-filtration HPLC [5, 61. The separation of PZP3a and PZP3p is only successful after the removal of sialylated and/or sulfated N-acetylpoly(1ac-tosamine) by digestion with endo-j-galactosidase 16, 71. We refer to the purified PZP3a and PZP3p as endo-p-galactosidasedigested PZP3a (EPG-PZP3a; 46 kDa) and endo-p-galactosidase-digested PZP3P (EpG-PZP3B; 42 kDa), respectively. The major component, PZP3, inhibited sperm-egg binding in an in vitro competition assay and E,b'G-PZP3a retained the inhibitory activity [8]. EpG-PZP3P showed no inhibition in this assay [ S ] . It has been reported that the minor component PZPl has also a small but significant inhibitory effect on sperm-egg binding in an in vitro competition assay 191. Using the fluorescein-labelled components of pig zona pellucida, the binding of PZP3 and EPG-PZP3a to boar sperm has been observed, but the binding of PZPl to boar sperm has not been detected [lo]. PZPl did not effectively inhibit the binding of biotinylated PZP3 to sperm membrane [ l 11. Thus, PZP3a may play a major role in sperm- egg binding and we have focused on the involvement of EPGPZP3a in sperm-egg binding in this study.Since the chemical deglycosylation of PZP3 with trifluoromethane sulfonate abolished its sperm receptor activity 18, 91, it is suggested that the sperm receptor activity depends upon the carbohydrate component of PZP3. The involvement of carbohydrates of zona pellucida in sperm-egg recognition has been well examined in mouse. It has been reported that the sperm receptor activity of mouse ZP3 (83 kDa...

The three glycoproteins of pig zona pellucida (ZPA, ZPB and ZPC) can be separated into ZPA and a mixture of ZPB/ZPC by gel-filtration HPLC. We have shown previously that the neutral complex-type N-linked carbohydrate chains obtained from ZPB/ZPC possess sperm-binding activity. Intact ZPB and ZPC cannot be separated from each other unless acidic N-acetyllactosamine regions of their carbohydrate chains are removed by endo-β-galactosidase digestion. The endo-β-galactosidaseϪdigested ZPB retains the sperm-binding activity. Recently, we have reported that N-linked carbohydrate chains of N-terminal fragment (residues 137Ϫ247) obtained from endo-β-galactosidaseϪdigested ZPB are involved mainly in sperm binding [Yonezawa, N., Mitsui, S., Kudo, K. & Nakano, M. (1997) Eur. J. Biochem. 248, 86Ϫ92]. In this study, we separated the intact neutral N-linked chains from the ZPB/ZPC mixture into diantennary chains and triantennary and tetraantennary chains by affinity chromatography on Concanavalia ensiformis agglutinin. An in vitro competition assay revealed that triantennary and tetraantennary chains possess a sperm-binding activity stronger than that of diantennary chains. Three glycopeptides, having one Asn residue to which the carbohydrate chain is linked, were obtained by lysyl endopeptidase digestion of the heat-solubilized zonae containing intact ZPB and lysyl endopeptidase and chymotrypsin A digestion of endo-β-galactosidaseϪdigested ZPB. From sugar-mapping analysis of the carbohydrate chains from these glycopeptides and comparison with the carbohydrate structures of the main intact neutral N-linked chains of ZPB/ZPC, the triantennary and tetraantennary chains were shown to be localized mainly at Asn220 of ZPB, and diantennary chains were present on all the three potential residues (Asn203, Asn220 and Asn333). These results suggest that the carbohydrate chains linked to Asn220 of ZPB participate predominantly in sperm-egg binding.Keywords : glycoprotein ; mammalian fertilization; N-linked carbohydrate chain; sperm ligand; zona pellucida.At the early phase of mammalian fertilization, sperm bind to tures of the O-linked chains that possess sperm-binding activity have not been elucidated. The sperm-binding region inhibits the carbohydrate chain(s) of zona pellucida glycoproteins in a species-specific manner [1,2]. Zona pellucida, which surrounds competitively sperm-egg binding. In pigs, it has been shown that the neutral N-linked carbohydrate chains obtained from the pig the mammalian oocyte, consists of three glycoproteins, ZPA, ZPB and ZPC, in the order of the molecular mass of the protein ZPB/ZPC mixture possess sperm-binding activity [8]. This neutral N-linked chain fraction is composed of di-, tri-, and tetraskeleton from high to low [3]. Pig ZPA, ZPB and ZPC are also called PZPL, PZP3A, and PZP3β, respectively [4Ϫ6]. ZPA and antennnary complex-type chains [8].The ORF of pig ZPB cDNA encodes a polypeptide of 536 the mixture of ZPB and ZPC (ZPB/ZPC mixture is also called PZP3) are separated by gel filtration [4], but Z...

beta-N-Acetylhexosaminidase (beta-Hex, EC, 3.2.1.52) was released from cauda epididymal boar sperm by treatment with ionophore A23187, indicating that this enzyme is localized in the acrosome. beta-Hex was extracted on a large scale, with 2% acetic acid containing 0.2% Brij 35, from washed ejaculated sperm. By gel filtration chromatography, beta-Hex was separated into a high-molecular-weight fraction (beta-Hex I) and a low-molecular-weight fraction (beta-Hex I). beta-Hex I, which is predominant under acidic conditions (pH 6.5), dissociated into beta-Hex II under alkaline conditions (pH 7.4). beta-Hex II, converted from beta-Hex I, associated again to form beta-Hex I under acidic conditions. By sequential chromatography on ion-exchange, lectin, gel filtration, and ion-exchange HPLC columns, beta-Hex I and II were purified 1200-fold and 4000-fold, respectively, with a combined recovery of 23% as measured with synthetic substrate. An inhibitor of beta-Hex, O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino N-phenyl-carbamate (PUGNAC), reduced the in vitro fertilization rate in porcine cumulus-enclosed eggs, but barely changed the rate when cumulus-free eggs were used. beta-Hex I was shown to possess cumulus dispersion activity, suggesting that beta-Hex plays a role in the passing by sperm through cumulus cells before they bind to the zona pellucida.

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