Takehiro Kawano

A major glycoprotein of Xenopus egg vitelline envelope, gp41, is a frog homolog of mammalian ZP3

A predominant glycoprotein in the vitelline envelope (VE) of the anuran Xenopus laevis is gp41, known to be proteolytically converted from gp43 of the coelomic egg envelope concomitant with the acquisition of egg fertilizability. To characterize the protein core of gp41, purified gp41 from VE was digested with lysyl endopeptidase, and peptides isolated from the digests were sequenced for amino acids to design degenerate primers for polymerase chain reaction. By reverse transcription‐polymerase chain reaction with a poly(A)+ RNA from the ovary of an ovulated female Xenopus, a specifically amplified band was obtained and sequenced. The upstream and downstream sequences of the sequenced region were completed by 5′‐ and 3′‐rapid amplification of cDNA ends, respectively. The cDNA, referred to as gp43 cDNA, comprises 1423 base pairs and contains one open reading frame with a sequence for 460 amino acids. The predicted amino acid sequence of gp43 cDNA has a close similarity with that of mammalian ZP3. Northern blot and in situ hybridization studies indicated that gp43 mRNA is expressed in oocytes, particularly in the previtellogenic oocytes. A comparison of the N‐terminal sequences of gp41 and gp43 strongly suggested that gp41 is generated at least by processing of the N‐terminal portion of gp43 with oviductin.

Structural differences found in the sugar chains of eutopic and ectopic free α-subunits of human glycoprotein hormone

Free alpha-subunits of human glycoprotein hormone were purified from the urine of a healthy pregnant woman and from that of a patient with adenocarcinoma. Comparative study of their sugar moieties revealed that they have different numbers and different sets of asparagine-linked sugar chains, which are also different from those of alpha-subunit obtained by dissociation of whole hCG molecule. The eutopic free alpha-subunit contained biantennary complex-type sugar chains only. In contrast, the ectopic free alpha-subunit contained tri- and tetraantennary complex-type sugar chains in addition.

A naturally occurring 46-amino acid deletion of cytidine monophospho-N-acetylneuraminic acid hydroxylase leads to a change in the intracellular distribution of the protein

Cytidine monophospho-N-acetylneuraminic acid (CMP-NeuAc) hydroxylase is a key enzyme for the expression ofN-glycolylneuraminic acid. The molecular cloning of this enzyme from mouse liver has been described in our previous report (Kawano T, Koyama S, Takematsu H, Kozutsumi Y, Kawasaki H, Kawashima S, Kawasaki T, Suzuki A (1995)J Biol Chem270: 16458–63). During the cDNA cloning, a cDNA containing a truncated open reading frame (ORF) was isolated. This clone encodes a protein of 531 amino acids which lacks 46 amino acids in the middle of the normal full-length protein. The percentage of this mRNA containing the truncated ORF out of the total population of CMP-NeuAc hydroxylase mRNA in various mouse tissues was about 10–25%. The truncated protein was expressed in COS-1 cells, but did not show any enzymatic activity. The truncated protein was localized to the region which appeared to be the endoplasmic reticulum, whereas the full-length protein with normal enzymatic activity was detected in the cytosol. These data suggest that this naturally occurring 46-amino acid deletion leads to a change in the intracellular distribution of CMP-NeuAc hydroxylase, and a loss in the activity of this enzyme.

S18.15 The key enzyme which regulates CMP-N-acetylneuraminic acid hydroxylation is a novel protein

determined as KDNa2o3Gal/31 ~4Glc/31 ~ 1 ceramide, (KDN)GM3 (1). Following this finding, two minor KDNgangliosides were isolated from ovarian fluid of rainbow trout and their structures were established by chemical degradation, methylation analysis and ~H NMR spectroscopy as (KDN)GD la and (9-O-AcKDN)GDla (2). In addition to these gangliosides, several other minor KDN-gangliosides have been identified. The results of these studies have suggested the general existence of KDN-gangliosides in a wider range of animal tissues and the failure to find them was apparently due to the methodology used. To search for KDN-gangliosides, we have set out to develop sensitive probes and availability of different types of KDNglycoconjugates has enabled us to generate monoclonal antibodies which specifically recognize KDN-glycan epitopes such as KDNa2~3Gal/31 ~ and KDNa2~8KDNa2 ~ sequences (3). With these sensitive probes (mAb.kdn3G and mAb. kdn8kdn), the rapid detection not only of major but also minor KDN-glycoconjugates should be feasible, and low relative molecular mass material extracted by chloroform/ methanol from various animal origins was tested for recognition by mAb.kdn3G and mAb.kdn8kdn. We have indeed preliminary evidence for the presence of mAb.kdn3G-positive antigens in some tissues. Our interest in KDN-gangliosides has been extended to the synthesis of neo-KDN-gangliosides, i.e., potentially useful analogues for the corresponding gangliosides, by transferring the KDN residue(s) from CMP-KDN to a variety of Neu5Acgangliosides and asialo-gangliosides with the aid of the KDNtransferase activities that we recently identified (4). (1) Yu Song, K. Kitajima, S. Inoue and Y. Inoue (1991) J. Biol. Chem., 266, 21929-21935; (2) Yu Song, K. Kitajima, S. Inoue, H. Muto, T. Kasama, S. Handa and Y. Inoue (1993) J. Biol. Chem. submitted; (3)Yu Song, K. Kitajima and Y. Inoue (1993) Glycobiology, 3, in press; (4)T. Terada, Yu Song, T. Angata, S. Kitazume, K. Kitajima, S. Inoue, F .A. Troy and Y. Inoue (1993) In the 12th International Symposium on Glycoconjugates.

S18.19 A ternary complex consisting of cytochrome B5, CMP-N-acetylneuraminic acid hydroxylase and its substrate is formed in the hydroxylation reaction

(Neu5Ac, Neu5Gc)~ separated by intervening segments of (Neu5Gc)n and (Neu5Ac)m of variable length depending on the particular brook trout PSGP subfraction, and the latter of which, i.e. (Neu5Ac)m would serve as epitopes for H.46 antibody. The Neu5Ac content in Salvelinus fish PSGPs ranges from 0 to 100%. Analysis of the structure of 8 PSGPs has thus shown that they can be classified into at least three groups on the basis of the PSA structures: (a) a-2~8-1inked poly(Neu5Ac)-containing PSGPs; (b) a-2~8-1inked poly(Neu5Ac,Neu5Gc)-containing PSGPs; (c) a-2-~8-1inked poly(Neu5Gc)-containing PSGPs. Therefore, they offer a model system in which to study the diversity of PSA structures of animal PSGPs. In addition to these PSA forms, we found recently another distinct type of PSA, i.e. a-2o8-1inked oligo/ poly(KDN) in the glycoprotein isolated from the vitelline envelope of rainbow trout. In summary, the present study demonstrates that PSGPs from salmonid fish species are oligo/polysialylated in a species-specific manner and appear to contain a-2-~8-1inked oligo/poly(Sia) with diverse structures. Work is in progress to explore the possible use of these PSGP molecules in immunization against various types of poly(Sia) residues to produce monoclonal antibodies specific to each form of PSA in order to provide useful probes for identifying such PSA structures in animal species other than fish.