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An aminopeptidase N (APN) with a molecular weight of 110kDa was released from the midgut membrane of Bombyx mori by phosphatidylinositol-specific phospholipase C (PI-PLC), and purified to a homogeneous state. This 110-kDa APN was different from the 100-kDa APN that we previously reported, in chromatographic behaviors, substrate specificity, and N-terminal and internal amino acid sequences. However, the N-terminal sequence of 110-kDa APN, DPAFRLPTTTRPRHYQVTLT, was highly homologous with those of Manduca sexta and Heliothis virescens APNs, which were identified as a receptor for an insecticidal toxin of Bacillus thuringiensis. From a B. mori midgut cDNA library, we cloned the 110-kDa APN cDNA that possessed a 2958-bp open reading frame encoding a 111573-Da polypeptide of 986 residues. The sequence of the eicosa-peptide Asp42Thr61 deduced from the cDNA was completely matched with the N-terminal sequence of the mature 110-kDa APN. One potential N-glycosylation site, HEXXHXW zinc-binding motif and characteristic proline-rich repeats were observed in the ORF. Moreover, the primary sequence contained two hydrophobic peptides on N- and C-termini. The N-terminal peptide sequence showed characteristics of leader peptide for secretion and the C-terminal peptide contained a possible glycosylphosphatidylinositol (GPI) anchoring site. Taken together, the deduced amino acid sequence suggests that the 110-kDa APN is a GPI-anchored protein and a specific receptor protein for B. thuringiensis CryIA delta-endotoxin.

We studied the effects of different types of dietary fats on fatty acid composition and activity of protein kinase C (PKC) in rat colonic mucosa. Activation of PKC, a key enzyme in signal transduction and growth regulation, provides a mechanism by which dietary components could be involved in colon carcinogenesis. Male Wistar rats (n = 12/group) were fed a semisynthetic high fat diet (43% of energy) containing either sunflower oil, rapeseed oil, or butter for 4 weeks ad libitum. The control group received a low fat sunflower oil diet (10% of energy). The butter diet increased membrane-associated PKC activity in rat colonic mucosa compared with the low fat control diet (1237 vs. 917 pmol/min per mg prot.; P = 0.028). Mucosal fatty acids reflected dietary fatty acid composition even though there was no clear association between the amount of mucosal fatty acids and PKC activity. More research is needed to elucidate how dietary fatty acids regulate colonic PKC activity.

A large glucose polymer was found, following pronase digestion, in PC12 pheochromocytoma cells metabolically labeled with [1-3H]galactose. The polymer was excluded from a Bio-Gel A-0.5 m column and adsorbed by immobilized concanavalin A-Sepharose from which it was eluted with 10 mM alpha-methylmannoside. Glucose was found to be the sole component monosaccharide. Except for those capable of degrading glycogen, no exo- or endo-glycosidases cleaved the polymer. This is the first report on the occurrence of a glucose polymer in undifferentiated PC12 cells.

Neutral glycolipids in PC12 cells were examined. A major neutral glycosphingolipid, isolated from a chloroform/methanol extract of the cells, was found to contain only galactose and glucose at a ratio of 3:1 and identified as ceramide tetrahexoside by fast atom bombardment (FAB) mass spectrometry. Its saccharide sequence was determined by a new method developed here using endoglycoceramidase (Ito, M., and Yamagata, T. (1986) J. Biol. Chem. 261, 14278-14282). The glycosphingolipid was digested with endoglycoceramidase to produce oligosaccharide which was subsequently pyridylaminated. The fluorescence-labeled oligosaccharide was digested with a series of specific exoglycosidases and fractionated by high performance liquid chromatography. The 2-aminopyridyl oligosaccharide was hydrolyzed by alpha-galactosidase to give a 2-aminopyridyl oligosaccharide which was identified as 2-aminopyridyl lactose by high performance liquid chromatography, indicating the glycolipid structure to be Gal alpha Gal alpha Gal beta GlcCer. Ceramide trihexoside obtained by limited digestion of the intact glycolipid was clearly identical with ceramide trihexoside obtained from human erythrocytes, according to NMR spectroscopy and methylation analysis. From these and other data on the intact glycolipid, obtained by methylation analysis and NMR spectroscopy, its structure was confirmed as Gal alpha 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer, III3-Gal alpha-globotriaosylceramide. This is the first report indicating the presence of this glycosphingolipid in PC12 cells.

Simultaneous separation of N-acetylglucosaminitol, N-acetylgalactosaminitol, N-acetylglucosamine and N-acetylgalactosamine from each other was achieved by paper chromatography. The method, which is suitable for analysis of radioactively labeled glycopeptides, allowed identification of N-acetylglucosamine and N-acetylgalactosamine as the linkage sugars in specific glycopeptide fractions isolated from human teratocarcinoma cells of line PA 1.

Human teratocarcinoma derived cells, line PA 1, maintained in the undifferentiated state, yielded upon exhaustive pronase digestion unusually large glycopeptides (fraction A), which showed on gel filtration an apparent molecular weight larger than 7400. These glycopeptides derived from whole cell proteins carried large-sized oligosaccharides as evidenced by repeated pronase treatments, hydrazinolysis, and beta-elimination experiments. The oligosaccharides consisted of mannose, fucose, galactose, and N-acetylglucosamine. The PA 1 cells contained also oligomannosyl type glycans, presumably linked to asparagine (fraction C glycopeptides). These glycopeptides were strongly bound to Con A--Sepharose and their oligosaccharides were released by endo-beta-N-acetylglucosaminidase H. The liberated glycans ranged from Man5GlcNAc to Man9GlcNAc as analyzed by paper chromatography. "Pulse-chase" experiments suggest that there is a precursor-product relationship between the mannose label in the fraction C (oligomannosyl type) glycopeptides and the fraction A glycopeptides.

Pieces of previously neurally induced and competent epiblast of chick and, respectively, quail primitive streak blastoderms were cultured in close contact with each other for 48 h. In several cases, both pieces differentiated into neural direction, which indicates the occurrence of a homoiogenetic induction. There was a considerable mixing of cells of different origin, especially in the undifferentiated controls. In general, the dorsoventral orientation of the previously induced epiblast was retained, but the orientation of the competent epiblast cells was more flexible and could be influenced by the neighbouring neuralised cells.

The glucose polymer of PC12 cells (Rasilo, M.-L. and Yamagata, T., 1988, FEBS Letters 227, 191-194 and Rasilo, M.-L. and Yamagata, T., 1988, Journal of Biochemistry, 104, 742-754) was found to be located on the cell surface. The polymer was liberated from the galactose-labeled cells with a trypsin treatment: maximally 65% of the glucose polymer was liberated, compared with 36% of the large glycopeptides. Even when the cells were incubated with the saline about one fourth of the polymer moved into the solution, but less than 8% of the large glycopeptides. Phosphatidyl inositol-specific phospholipase C failed to liberate the polymer.

Pronase digests of cultured teratocarcinoma-derived cells (PA 1) of human origin have been previously shown to contain large-sized glycopeptides (relative mass (Mr) greater then 7400), of which 15-23% are retained by columns of concanavalin A (Con A) - Sepharose and can be eluted with 10 mM methyl alpha-D-mannopyranoside. The present data show that this fraction (A - Con A II) contains a family of glycopeptides that are degradable with anhydrous hydrazine as well as with 0.05 M NaOH - 1 M NaBH4. The cleavage products representing individual oligosaccharide chains, presumably as oligosaccharides and glycopeptides, consisted mostly of medium- (Mr 1400-6000) and small-sized (Mr less than 1400) molecules. This implies that glycopeptides bearing several oligosaccharide chains were present in A - Con A II. Most of the individual oligosaccharide chains were not bound to Con A - Sepharose, but some were retained by the lectin column in the same way as the original glycopeptides. Some of the oligosaccharides were degraded partially with endo-beta-galactosidase from Escherichia freundii suggesting the presence of GalbetaGlcNAcbeta repeats. The present findings show that A - Con A II may be different from the "embryonic" glycopeptides of mouse teratocarcinoma cells that are reportedly not cleaved by mild alkaline borohydride treatment. Instead, A - Con A II is reminiscent of the T-1 glycopeptide of glycophorin.

The glycans of well characterized, [6-3H]galactose-labelled glycopeptides, GC-4 from bovine IgG1 as well as GP-V-2 and GP-V-5 from alpha1-acid glycoprotein, were liberated by hydrazinolysis. Molecular weights close to the expected values were observed by gel filtration. Desialated glycans of Semliki Forest virus proteins were likewise liberated by hydrazinolysis and subjected to gel filtration. Metabolically labelled [1-3H]galactose-oligosaccharides of the mixed viral proteins revealed an apparent molecular weight of 1800. The bi-antennary glycan liberated from the reference glycopiptide GC-4 was of 1750 daltons. A mixture of [2-3H]mannose-labelled E1- and E2-proteins of the virus contained L-type glycans of 1800 daltons (formerly called A-type), and M-type glycans of 1200 daltons (formerly called B-type). A fraction of the E3-glycans isolated by affinity chromatography on Concanavlin A-Sepharose showed an average molecular weight of 2150, a value intermediate between the three- and four-antennary glycans liberated from the reference glycopeptides GP-V-5 and GP-V-2. The rest of the E3-glycans were of 1850 daltons, a value close to the bi-antennary GC-4 glycan. We suggest that the comparatively large size of the E3-glycans and the exposed position of E3-proteins on the viral surface may be interrelated.

Endoglycoceramidase (EGCase) cleaves the linkage between the oligosaccharide and ceramide of glycosphingolipids (Ito, M., and Yamagata, T., J. Biol. Chem. 261, 14278-14282, ibid, 264, in press). Intact cells of rat pheochromocytoma line PC12 were treated with the highly purified EGCase I and the oligosaccharides released were analyzed by HPLC. Cleavage of the oligosaccharides with the enzyme reached a plateau as the amount of the enzyme was increased. At maximum, 42% of the oligosaccharides from globoside, 40% from GalGb3Cer, and 60% from Gb3Cer were liberated during 1h of incubation without impairing the viability of cells. The only partial liberation indicates that not all oligosaccharides of cell surface glycosphingolipids are accessible to the enzyme. The use of EGCase offers an important new method to access the functions of glycosphingolipids on cell surface in situ.

N-acetyl-lactosamine type glycopeptides of Semliki Forest virus were fractionated on concanavalin A-Sepharose, and their oligosaccharides were liberated by hydrazinolysis and analyzed by paper chromatography. Comparison with labeled glycans from reference glycopeptides suggests that the viral N-acetyl-lactosamine type glycans are bi-, tri- and tetra-antennary.

Human teratocarcinoma-derived cells of line PA 1, which are capable of differentiating in vitro [Zeuthen, J. et al. (1980) Int J. Cancer, 25, 19-32], incorporate label from radioactive sulfate and/or glucosamine into several large-sized glycosaminoglycans including hyaluronate, chondroitin sulfate/dermatan sulfate co-polymers, heparan sulfate and keratan-sulfate-like molecules. All these polysaccharide fractions were identified by specific degradation methods. The labeled hyaluronate was degraded into a mixture of unsaturated octa-, hexa- and tetra-saccharides by a treatment with Streptomyces hyaluronidase (EC 4.2.2.1). The chondroitin sulfate/dermatan sulfate co-polymers were cleaved with chondroitin AC lyase (EC 4.2.2.5) into unsaturated disaccharides and a series of unsaturated oligosaccharides; the latter were degraded by a treatment with chondroitin ABC lyase (EC 4.2.2.4) into unsaturated disaccharides. Heparan sulfate was degraded with nitrous acid into free inorganic [35S]sulfate and a series of [35S]sulfate-labeled oligosaccharides and/or glycopeptides. The keratan-sulfate-like molecules were hydrolyzed by a treatment with endo-beta-galactosidase from Escherichia freundii into a series of distinct [35S]sulfate-labeled oligosaccharides; small oligosaccharides were liberated also from [3H]galactose-labeled molecules. The smallest one of the liberated oligosaccharides was tentatively identified as a sulfated disaccharide.

Labelled oligomannosyl type glycans of E2 protein of Semliki Forest virus were liberated by treatment with endo-beta-N-acetylglucosaminidase H, as well as by hydrazinolysis and analysed by paper chromatography. The protein-bound virus oligosaccharides were a mixture of Man7GlcNAc2, Man6GlcNAc2 and Man5GlcNAc2, and Man8GlcNAc2 also appeared to be present. P62 protein, the direct precursor of E2, was labelled by supplying infected BHK cells with 2-3H-mannose for 5 min. The oligomannosyl glycans of the P62 proteins formed were about two monosaccharide units larger than those of the mature virus E2 proteins.

A large-sized glucose polymer was isolated by pronase digestion from line PC12 pheochromocytoma cells metabolically labeled with [1-3H]galactose. The polymer was included on a column of concanavalin A-Sepharose and could be eluted with 10 mM methyl-alpha-mannoside. Its slight retention in a column of Bio-Gel A-5m suggested that its molecular weight was in the several millions. Glucose was the component monosaccharide and there were two minor lipophilic components present. The polymer was digested with alpha-amylase into a series of oligosaccharides and was cleaved by glucoamylase into glucose residues. The disaccharide obtained by digestion with alpha-amylase was identified as maltose in several HPLC systems and by NMR spectroscopy. NMR measurement revealed the trisaccharide to be maltotriose. Susceptibility of the polymer molecule to alpha-amylase, and the digestion products obtained, indicated a resemblance to glycogen. An analysis for saccharide compositions before and after reduction of the polymer suggested the presence of an aglycon part. Contrary to expectations based on the presence of this moiety, the polymer displayed good solubility in neutral organic solvents. Two-thirds of the glucose polymer was also soluble in 10% TCA. A similar glucose polymer was isolated from neuronal cells of rat embryos metabolically labeled with [1-3H]galactose. Mouse neuroblastoma cells did not synthesize the polymer.

Human teratocarcinoma derived cells, line PA 1, were labeled with radioactive monosaccharides and subsequently digested with pronase. Large sized glycopeptides (fraction A) were isolated by gel filtration on Bio-Gel P-10. Their chromatography on concanavalin A-Sepharose gave three subfractions, two of which were eluted with a sugar-free buffer and the third with 10 mM alpha-methyl mannoside. The first subfraction (fraction A-Con A Ia) incorporated label from [3H]galactose and [3H]glucosamine and contained the largest components of fraction A. The second and the third subfractions (fractions A-Con A Ib and A-Con A II) were glycopeptides which incorporated label from tritiated fucose, mannose, galactose, and glucosamine. Even these molecules were of large size eluting partially at the void volume from Bio-Gel P-60. The glycopeptides of fraction A-Con A Ib contained mannose, fucose, galactose, N-acetylglucosamine, and N-acetylgalactosamine. Fucose and galactose residues occupied ultimate or penultimate positions at the nonreducing termini of the oligosaccharides. N-Acetylneuraminic acid, too, was present in the glycopeptides of fraction A.