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Biomedical subjects

Ken Kitajima

Publications and source records attributed to Ken Kitajima.

At least 19 recordsLinked to original sources
PubMed2006

Complex formation of 70-kDa heat shock protein with acidic glycolipids and phospholipids.

A new property of a heat-inducible heat shock protein (Hsp) 70.1 that it forms a complex with acidic lipids was first demonstrated. Based on the behaviors of the complexes on the native PAGE, the acidic lipid/Hsp70.1 complexes are categorized into two groups. The first group is the sulfatide-induced large-sized complex, which stays on the gel top on the native PAGE. Only the N-terminal ATPase domain is responsible for the complex formation. The second group is the ganglioside-induced complex, which is diffused in the resolution gel on the native PAGE. Both the N-terminal ATPase and the C-terminal peptide-binding domains are involved in the complex formation. No complex is formed by neutral glyco- and phospholipids. The complex formation with the acidic glyco- and phospholipids implicates the various functions of Hsp70 on the membrane surfaces.

Animals↗
PubMed2006

Involvement of the alpha2,8-polysialyltransferases II/STX and IV/PST in the biosynthesis of polysialic acid chains on the O-linked glycoproteins in rainbow trout ovary.

Polysialoglycoprotein (PSGP) in salmonid fish egg is a unique glycoprotein bearing alpha2,8-linked polysialic acid (polySia) on its O-linked glycans. Biosynthesis of the polySia chains is developmentally regulated and only occurs at later stage of oogenesis. Two alpha2,8-polysialyltransferases (alpha2,8-polySTs), PST (ST8Sia IV) and STX (ST8Sia II), responsible for the biosynthesis of polySia on N-glycans of glycoproteins, are known in mammals. However, nothing has been known about which alpha2,8-polySTs are involved in the biosynthesis of polySia on O-linked glycans in any glycoproteins. We thus sought to identify cDNA encoding the alpha2,8-polyST involved in polysialylation of PSGP. A clone for PST orthologue, rtPST, and two clones for the STX orthologue, rtSTX-ov and rtSTX-em, were identified in rainbow trout. The deduced amino acid sequence of rtPST shows a high identity (72-77%) to other vertebrate PSTs, while that of rtSTX-ov shows 92% identity with rtSTX-em and a significant identity (63-76%) to other vertebrate STXs. The rtPST exhibited the in vivo alpha2,8-polyST activity, although its in vitro activity was low. However, the rtSTXs showed no in vivo and very low in vitro activities. Interestingly, co-existence of rtPST and rSTX-ov in the reaction mixture synergistically enhanced the alpha2,8-polyST activity. During oogenesis, rtPST was constantly expressed, while the expression of rtSTX-ov was not increased until polySia chain is abundantly biosynthesized in the later stage. rtSTX-em was not expressed in ovary. These results suggest that the enhanced expression of rtSTX-ov under the co-expression with rtPST may be important for the biosynthesis of polySia on O-linked glycans of PSGP.

Amino Acid Sequence↗
PubMed2006

Flagellasialin: a novel sulfated alpha2,9-linked polysialic acid glycoprotein of sea urchin sperm flagella.

A novel alpha2,9-linked polysialic acid (polySia)-containing glycoprotein of sea urchin sperm flagella was identified and named "flagellasialin." Flagellasialin from Hemicentrotus pulcherrimus shows a diverse relative molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of 40-80 kDa. Flagellasialin is a 96-amino acid, threonine-rich, heavily O-glycosylated (80-90% by weight) glycoprotein with a single transmembrane segment at its C-terminus and no apparent cytosolic domain. Of 12 extracellular Thr residues, eight are O-glycosylated and three are nonglycosylated. Flagellasialin is highly expressed in the testis but cannot be detected in the ovary. The amino acid sequences of flagellasialin from three sea urchin species (H. pulcherrimus, Strongylocentrotus purpuratus, and Strongylocentrotus franciscanus) are identical, but some species differences exist in the three core glycan structures to which the sulfated alpha2,9-linked polyNeu5Ac chain is linked. Finally, the treatment of sperm with a specific antibody against the alpha2,9-linked polyNeu5Ac structure results in the elevation of intracellular Ca(2+) and inhibition of sperm motility and fertilization, implicating flagellasialin as a regulator of these critical processes.

Amino Acid Sequence↗
PubMed2006

Complete prevention of atherosclerosis in apoE-deficient mice by hepatic human apoE gene transfer with adeno-associated virus serotypes 7 and 8.

OBJECTIVE: Using intravenous injection of adeno-associated viral (AAV) vectors based on novel serotypes 7 and 8, we examined whether liver-specific expression of human apolipoprotein E (apoE) in apoE-deficient mice would completely prevent atherosclerosis after 1 year of sustained expression. METHODS AND RESULTS: Chow-fed apoE-/- mice were injected via the tail vein with vectors based on AAV2 or novel serotypes AAV7 and AAV8 encoding human apoE3 driven by a liver-specific promoter. In contrast to the first-generation AAV2 vector, apoE levels of mice injected with chimeric AAV2/7 and AAV2/8 vectors reached approximately 2-fold greater than normal human plasma levels by week 4 and maintained therapeutic levels up to 1 year. Cholesterol levels of AAV2/7-apoE and AAV2/8-apoE-treated mice were reduced to normal murine wild-type levels and were maintained for 1 year. At termination after 1 year, extensive atherosclerosis was present in the thoracic aortas and aortic roots of control AAV2/8-lacZ and AAV2-apoE-injected mice, but was completely prevented in both the AAV2/7 and AAV2/8-apoE-treated mice. CONCLUSIONS: We demonstrate that intravenous administration of AAV2/7- and AAV2/8-apoE vectors effectively mediated robust and sustained hepatic-specific expression of apoE and completely prevented atherosclerosis at 1 year.

Animals↗
PubMed2006

N-linked oligosaccharides on chondroitin 6-sulfotransferase-1 are required for production of the active enzyme, Golgi localization, and sulfotransferase activity toward keratan sulfate.

We have shown previously that purified chondroitin 6-sulfotransferase-1 (C6ST-1) was a glycoprotein abundant in N-linked oligosaccharides and could sulfate both chondroitin (C6ST activity) and keratan sulfate (KSST activity); however, functional roles of the N-glycans have remained unclear. In the present study, we show essential roles of N-glycans attached to C6ST-1 in the generation of the active enzyme and in its KSST activity. Treatment with tunicamycin of COS-7 cells transfected with C6ST-1 cDNA totally abolished production of the active C6ST-1. A nearly complete removal of N-glycans of the recombinant C6ST-1 by peptide N-glycosidase F increased the C6ST activity but decreased the KSST activity. Among six potential N-glycosylation sites, deletion of the fourth or sixth site from the amino terminus inhibited production of the active C6ST-1, whereas deletion of the fifth site resulted in a marked loss of the KSST activity. Wild-type recombinant C6ST-1 showed a typical Golgi localization, whereas M-4 recombinant C6ST-1, in which the fourth N-glycosylation site was deleted, colocalized with calnexin, an endoplasmic reticulum-resident protein. Unlike wildtype recombinant C6ST-1, M-4 recombinant C6ST-1 showed a weak affinity toward wheat germ agglutinin and was converted completely to the nonglycosylated form by endoglycosidase H. These observations suggest that N-glycan attached to the fourth N-glycosylation site may function in the proper processing of N-glycans required for the Golgi localization, thereby causing the production of the active C6ST-1, and that N-glycan attached to the fifth N-glycosylation site may contribute to the KSST activity of C6ST-1.

Amino Acid Sequence↗
PubMed2006

Identification of disialic acid-containing glycoproteins in mouse serum: a novel modification of immunoglobulin light chains, vitronectin, and plasminogen.

Serum glycoproteins are involved in various biologic activities, such as the removal of exogenous antigens, fibrinolysis, and metal transport. Some of them are also useful markers of inflammation and disease. Although the amount of sialic acid increases following inflammation, little attention has been paid to the presence of linkage-specific epitopes in serum, especially the alpha2,8-linkage. In a previous study, we demonstrated that four components in mouse serum contain alpha2,8-linked disialic acid (diSia), based on immunoreactivity with monoclonal antibody 2-4B, which is specific to N-glycolylneuraminic acid (Neu5Gc)alpha2-->(8Neu5Gc alpha2-->)(n-1), n > or = 2 [Yasukawa et al., (2005) Glycobiology, 15, 827-837]. In this study, we purified three components, 30-, 70-, and 120-kDa gp, and identified them as an immunoglobulin (Ig) light chain, vitronectin, and plasminogen, respectively, using matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy analyses. Modifications of these proteins with alpha2,8-linked diSia were chemically confirmed by fluorometric C7/C9 analyses and mild acid hydrolysates-fluorometric anion-exchange chromatography analyses. We also demonstrated that the IgG, IgM, and IgE light chains are commonly modified with alpha2,8-linked diSia. In addition, both mouse and rat vitronectin contained diSia, and the amount of disialylation in vitronectin dramatically decreased after hepatectomy. These results indicate that a novel diSia modification of serum glycoproteins is biologically important for immunologic events and fibrinolysis.

Amino Acid Sequence↗
PubMed2006

Hypoxic culture induces expression of sialin, a sialic acid transporter, and cancer-associated gangliosides containing non-human sialic acid on human cancer cells.

Tumor hypoxia figures heavily in malignant progression by altering the intracellular glucose metabolism and inducing angiogenic factor production, thus, selecting and expanding more aggressive cancer cell clones. Little is known, however, regarding hypoxia-induced antigenic changes in cancers. We investigated the expression of N-glycolyl sialic acid (NeuGc)-G(M2), a cancer-associated ganglioside containing non-human sialic acid, NeuGc, in human cancers. Cancer tissues prepared from patients with colon cancers frequently expressed NeuGc-G(M2), whereas it was virtually absent in nonmalignant colonic epithelia. Studies on cultured cancer cells indicated that the non-human sialic acid was incorporated from culture medium. Hypoxic culture markedly induced mRNA for a sialic acid transporter, sialin, and this accompanied enhanced incorporation of NeuGc as well as N-acetyl sialic acid. Transfection of cells with sialin gene conferred accelerated sialic acid transport and induced cell surface expression of NeuGc-G(M2). We propose that the preferential expression of NeuGc-G(M2) in cancers is closely associated with tumor hypoxia. Hypoxic culture of tumor cells induces expression of the sialic acid transporter, and enhances the incorporation of non-human sialic acid from the external milieu. A consequence of this is the acquisition of cancer-associated cell surface gangliosides, typically G(M2), containing non-human sialic acid (NeuGc), which is not endogenously synthesized through CMP-N-acetyl sialic acid hydroxylase because humans lack the gene for the synthetic enzyme. As hypoxia is associated with diminished response to radiotherapy and chemotherapy, NeuGc-G(M2) is a potential therapeutic target for hypoxic cancer cells.

Caco-2 Cells↗
PubMed2006

KDN (deaminated neuraminic acid): dreamful past and exciting future of the newest member of the sialic acid family.

KDN is an abbreviation for 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid, and its natural occurrence was revealed in 1986 by a research group including the present authors. Since sialic acid was used as a synonym for N-acylneuraminic acid at that time, there was an argument if this deaminated neuraminic acid belongs to the family of sialic acids. In this review, we describe the 20 years history of studies on KDN (KDNology), through which KDN has established its position as a distinct member of the sialic acid family. These studies have clarified that: (1) KDN occurs widely among vertebrates and bacteria similar to the occurrence of the more common sialic acid, N-acetylneuraminic acid (Neu5Ac), but its abundant occurrence in animals is limited to lower vertebrates. (2) KDN is found in almost all types of glycoconjugates, including glycolipids, glycoproteins and capsular polysaccharides. (3) KDN residues are linked to almost all glycan structures in place of Neu5Ac. All linkage types known for Neu5Ac; alpha2,3-, alpha2,4-, alpha2,6-, and alpha2,8- are also found for KDN. (4) KDN is biosynthesized de novo using mannose as a precursor sugar, which is activated to CMP-KDN and transferred to acceptor sugar residues. These reactions are catalyzed by enzymes, some of which preferably recognize KDN, but many others prefer Neu5Ac to KDN. In addition to these basic findings, elevated expression of KDN was found in fetal human red blood cells compared with adult red blood cells, and ovarian tumor tissues compared with normal controls. KDNase, an enzyme which specifically cleaves KDN-linkages, was discovered in a bacterium and monoclonal antibodies that specifically recognize KDN residues in KDNalpha2,3-Gal- and KDNalpha2,8-KDN-linkages have been developed. These have been used for identification of KDN-containing molecules. Based on past basic studies and variety of findings, future perspective of KDNology is presented.

Animals↗
PubMed2006

Oral ingestion of mannose alters the expression level of deaminoneuraminic acid (KDN) in mouse organs.

Deaminoneuraminic acid (KDN) is a unique member of the sialic acid family. We previously demonstrated that free KDN is synthesized de novo from mannose as its precursor sugar in trout testis, and that the amount of intracellular KDN increases in mouse B16 melanoma cells cultured in mannose-rich media [Angata et al. (1999) J. Biol. Chem. 274, 22949-56; Angata et al. (1999) Biochem. Biophys. Res. Commun. 261, 326-31]. In the present study, we first demonstrated a mannose-induced increase in intracellular KDN in various cultured mouse and human cell lines. These results led us to examine whether KDN expression in mouse organs is altered by exogenously administered mannose. Under normal feeding conditions, intracellular free KDN was present at very low levels (19-48 pmol/mg protein) in liver, spleen, and lung, and was not detected in kidney or brain. Oral ingestion of mannose, both short-term (90 min) and long-term (2 wk), resulted in an increase of intracellular KDN up to 60-81 pmol/mg protein in spleen and lung and 6.9-18 pmol/mg protein in kidney and brain; however, no change was observed in liver. The level of KDN in organs appears not to be determined only by the KDN 9-phosphate synthase activity, but might also be affected by other enzymes that utilize mannose 6-phosphate as a substrate as well as the enzymes that breakdown KDN, like KDN-pyruvate lyase. In blood, the detectable amount of free KDN did not change on oral ingestion of mannose. These findings indicate that mannose in the diet affects KDN metabolism in various organs, and provide clues to the mechanism of altered KDN expression in some tumor cells and aged organs.

Administration, Oral↗
PubMed2005

Persistent liver expression of murine apoA-l using vectors based on adeno-associated viral vectors serotypes 5 and 1.

Plasma levels of high-density lipoprotein-cholesterol (HDL-C) and apolipoprotein A-l (apoA-l) are inversely related to risk for coronary heart disease. Overexpression of apoA-l inhibits atherosclerosis in animal models. A method of stably expressing apoA-l using somatic gene transfer would be of interest. Pseudotyped adeno-associated virus (AAV) vectors comprised of inverted terminal repeats from AAV serotype 2 have been used for liver-directed gene transfers. We hypothesized that liver-directed gene transfer of apoA-l using vectors based on AAV serotypes 1 and 5 would result in higher-level, prolonged expression of apoA-l and increased HDL-C. To test this hypothesis we injected apoA-l-/- mice via the tail vein with either AAV2, AAV1 or AAV5 vectors encoding the murine apoA-l cDNA driven by the liver-specific thyroxine binding globulin promoter. Plasma levels of murine apoA-l and HDL-C were highest in mice injected with the AAV1-based vector and lowest in mice injected with the AAV2-based vector. Expression of apoA-l was stable up to 1 year after vector injection. These results indicate that AAV5 and AAV1 are more effective vectors for achieving higher levels of stable transgene expression of apoA-l after liver-directed gene transfer than AAV2. Furthermore, AAV1-based vectors generate higher apoA-l levels than AAV5-based vectors. It is possible that the levels of expression achieved using these vectors will be therapeutic in preventing atherosclerosis.

Animals↗
PubMed2005

N-linked oligosaccharides are required to produce and stabilize the active form of chondroitin 4-sulphotransferase-1.

C4ST-1 (chondroitin 4-sulphotransferase-1) transfers sulphate to position 4 of N-acetylgalactosamine in chondroitin. We showed previously that purified C4ST-1 from the culture medium of rat chondrosarcoma cells was a glycoprotein containing approx. 35% N-linked oligosaccharides. In the present paper, we investigated the functional role of the N-linked oligosaccharides attached to C4ST-1. We found that (i) treatment of recombinant C4ST-1 with peptide N-glycosidase F caused a marked decrease in activity, (ii) production of the active form of C4ST-1 by COS-7 cells transfected with cDNA of C4ST-1 was inhibited by tunicamycin, (iii) deletion of the N-glycosylation site located at the C-terminal region of C4ST-1 abolished activity, (iv) attachment of a single N-glycan at the C-terminal region supported production of the active form of C4ST-1, but the resulting recombinant enzyme was much more unstable at 37 degrees C than the control recombinant protein, and (v) truncation of C-terminal region up to the N-glycosylation site at the C-terminal region resulted in total loss of activity. These observations strongly suggest that N-linked oligosaccharides attached to C4ST-1 contribute to the production and stability of the active form of C4ST-1. In addition, the N-linked oligosaccharide at the C-terminal region appears to affect the glycosylation pattern of recombinant C4ST; a broad protein band of the wildtype protein resulting from microheterogeneity of N-linked oligosaccharides disappeared and four discrete protein bands with different numbers of N-linked oligosaccharides appeared when the N-linked oligosaccharide at the C-terminal region was deleted.

Animals↗
PubMed2005

Inflammation-dependent changes in alpha2,3-, alpha2,6-, and alpha2,8-sialic acid glycotopes on serum glycoproteins in mice.

The expression of acute-phase serum proteins increases in response to inflammatory stimuli. Most of these proteins are glycoproteins that often contain sialic acids (Sia). It is unknown, however, how the expression of Sia in these glycoproteins changes during inflammation. This study demonstrates changes in the alpha2,3-, alpha2,6-, and alpha2,8-Sia glycotopes on serum glycoproteins in response to turpentine oil-induced inflammation, based on lectin- and immunoblot analyses by using sialyl linkage-specific lectins, Maackia amurensis for the alpha2,3-Sia glycotope and Sambucus sieboldiana for the alpha2,6-Sia glycotopes, and monoclonal antibody 2-4B (mAb.2-4B) recognizing the di- and oligomers of the alpha2,8-Neu5Gc residue. There was an increase in a limited number of sialoglycoproteins containing the alpha2,3-, alpha2,6-, or alpha2,8-Sia glycotopes. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of the expression profiles of mRNAs for the known sialyltransferases in mouse liver during inflammation indicated the up-regulated expression of beta-galactoside alpha2,3-sialyltransferases (ST3Gal I and ST3Gal III) and beta-N-acetylgalactosaminide alpha2,6-sialyltransferase (ST6GalNAc VI) as well as beta-galactoside alpha2,6-sialyltransferase (ST6Gal I) mRNAs. Notably, ST3Gal I and III and ST6GalNAc VI are involved in the synthesis of the alpha2,3- and alpha2,6-Sia glycotopes on O-glycan chains and possibly on gangliosides, whereas ST6Gal I is specific for N-glycan chains. These results provide evidence for the inflammation-induced expression of sialyl glycotopes in serum glycoproteins. We demonstrated that inflammation significantly increased the expression of an unknown 32-kDa glycoprotein containing the alpha2,8-Sia glycotope. The mechanism for the increase in glycoprotein in inflamed mouse serum remains to be examined, as mRNA expression for all of the alpha2,8-sialyltransferases (ST8Sia I-VI) was unchanged during inflammation.

Acute-Phase Proteins↗
PubMed2005

Probing a CMP-Kdn synthetase by 1H, 31P, and STD NMR spectroscopy.

CMP-Kdn synthetase catalyses the reaction of sialic acids (Sia) and cytidine-5'-triphosphate (CTP) to the corresponding activated sugar nucleotide CMP-Sia and pyrophosphate PP(i). STD NMR experiments of a recombinant nucleotide cytidine-5'-monophosphate-3-deoxy-d-glycero-d-galacto-nonulosonic acid synthetase (CMP-Kdn synthetase) were performed to map the binding epitope of the substrate CTP and the product CMP-Neu5Ac. The STD NMR analysis clearly shows that the anomeric proton of the ribose moiety of both investigated compounds is in close proximity to the protein surface and is likely to play a key role in the binding process. The relative rates of the enzyme reaction, derived from (1)H NMR signal integrals, show that Kdn is activated at a rate 2.5 and 3.1 faster than Neu5Ac and Neu5Gc, respectively. Furthermore, proton-decoupled (31)P NMR spectroscopy was successfully used to follow the enzyme reaction and clearly confirmed the appearance of CMP-Sia and the inorganic pyrophosphate by-product.

Catalysis↗
PubMed2005

Development of a simple and efficient method for assaying cytidine monophosphate sialic acid synthetase activity using an enzymatic reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide converting system.

A new reliable method to assay the activity of cytidine monophosphate sialic acid (CMP-Sia) synthetase (CSS) has been developed. The activation of sialic acids (Sia) to CMP-Sia is a prerequisite for the de novo synthesis of sialoglycoconjugates. In vertebrates, CSS has been cloned from human, mouse, and rainbow trout, and the crystal structure has been resolved for the mouse enzyme. The mouse and rainbow trout enzyme have been compared with respect to substrate specificity, demonstrating that the mouse enzyme exhibits a pronounced specificity for N-acetylneuraminic acid (Neu5Ac), while the rainbow trout CSS is equally active with either of three Sia species, Neu5Ac, N-glycolylneuraminic acid (Neu5Gc), and deaminoneuraminic acid (KDN). However, molecular details that explain the pronounced substrate specificities are unknown. Understanding the catalytic mechanisms of these enzymes is of major importance, since CSSs play crucial roles in cellular sialylation patterns and thus are potential drug targets in a number of pathophysiological situations. The availability of the cDNAs and the obtained structural data enable rational approaches; however, these efforts are limited by the lack of a reliable high-throughput assay system. Here we describe a new assay system that allows product quantification in a reduced nicotinamide adenine dinucleotide (NADH)-dependent color reaction. The activation reaction catalyzed by CSS, CTP+Sia-->CMP-Sia+pyrophosphate, was evaluated by a consumption of Sia, which corresponds to that of NADH on the following two successive reactions: (i) Sia-->pyruvate+ManNAc (or Man), catalyzed by a sialic acid lyase (SAL), and (ii) pyruvate+NADH-->lactate+oxidized nicotinamide adenine dinucleotide (NAD+), catalyzed by a lactate dehydrogenase (LDH). Consumption of NADH can be photometrically monitored on a microtiter plate reader for a number of test samples at the same time. Furthermore, based on the quantification of CSS used in the SAL/LDH assay, relative activities toward Sia derivatives have been obtained. The preference of mouse CSS toward Neu5Ac and the ability of the rainbow trout enzyme to activate both KDN and Neu5Ac were confirmed. Thus, this simple and time-saving method is suitable for a systematic comparison of enzyme activity of structurally mutated enzymes based on the relative specific activity.

Animals↗
PubMed2005

Distal myopathy with rimmed vacuoles: impaired O-glycan formation in muscular glycoproteins.

Distal myopathy with rimmed vacuoles (DMRV), is an autosomal recessive disorder with early adult onset, displays distal dominant muscular involvement and is characterized by the presence of numerous rimmed vacuoles in the affected muscle fibers. The pathophysiology of DMRV has not been clarified yet, although the responsible gene was identified as that encoding UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase involved in the biosynthesis of sialic acids. To identify defective carbohydrate moieties of muscular glycoproteins from DMRV patients, frozen skeletal muscle sections from seven patients with DMRV, as well as normal and pathological controls, were treated with or without sialidase or N-glycosidase F followed by lectin staining and lectin blotting analysis. The sialic acid contents of the O-glycans in the skeletal muscle specimens from the DMRV patients were also measured. We found that Arachis hypogaea agglutinin (PNA) lectin reacted strongly with sarcolemmal glycoproteins in the DMRV patients but not with those in control subjects. alpha-Dystroglycan from the DMRV patients strongly associated with PNA lectin, although that from controls did not. The sialic acid level of the O-glycans in the DMRV muscular glycoproteins with molecular weights of 30 to 200 kd was reduced to 60 to 80% of the control level. The results show that impaired sialyl O-glycan formation in muscular glycoproteins, including alpha-dystroglycan, occurs in DMRV.

Adolescent↗
PubMed2004

A newly identified zona pellucida glycoprotein, ZPD, and dimeric ZP1 of chicken egg envelope are involved in sperm activation on sperm-egg interaction.

Fertilization begins with interaction between the sperm and the egg. The surface of the vertebrate oocyte is covered with the egg envelope, which is composed of ZP (zona pellucida) glycoproteins. We have identified two glycoproteins, ZP1/gp97 and ZPC/gp42, as the major components of the chicken egg envelope. In the present study, another 42 kDa protein, designated ZPD, has been found as a new major component of the chicken egg envelope. ZPD was specifically released from the egg envelope by ultrasonication treatment without urea. ZPD cDNA was cloned using a chicken granulosa cell cDNA pool. The deduced amino acid sequence showed that preproprotein of ZPD is composed of 418 amino acid residues with four potential N-glycosylation sites and includes a ZP domain, common in vertebrate ZP glycoproteins, and a transmembrane domain. ZPD belongs phylogenetically to a distinct group from known ZP glycoprotein subfamilies, ZPA, ZPB, and ZPC. In two-dimensional gel electrophoresis ZPD proteins were identified to be several isoforms with different pI values between 5 and 7. ZP1, ZPC and the newly identified ZPD were confirmed to be the major components of chicken egg envelope by MS of proteolytic digests of whole egg envelope. The in vitro incubation of chicken sperm with calcium ionophore A23187 induced sperm activation, resulting in the fragmentation and release of a 41 kDa PNA (peanut agglutinin)-positive glycoprotein and the decrease or loss of sperm PNA-stainability. The incubation with ZPD and dimeric ZP1, but not ZPC and monomeric ZP1, also induced the decrease or loss of sperm PNA-stainability, suggesting the in vitro sperm activation by these ZP components. Collectively, ZPD might bind loosely to egg envelope matrix and play a key role in the sperm activation on avian sperm-egg interaction.

Amino Acid Sequence↗
PubMed2004

A major flagellum sialoglycoprotein in sea urchin sperm contains a novel polysialic acid, an alpha2,9-linked poly-N-acetylneuraminic acid chain, capped by an 8-O-sulfated sialic acid residue.

A new type of polysialic acid (polySia) structure was demonstrated to occur in a major unknown sialoglycoprotein with a diverse molecular mass of 40-80 kDa in sea urchin sperm. The polySia-containing glycan structure was determined to be HSO(3)-->8Neu5Acalpha2-->9(Neu5Acalpha2-->9)(n-2) Neu5Acalpha2-->6GalNAcalpha1-->Ser/Thr (n, on average 15), based on carbohydrate analysis of the sialoglycopeptide obtained by an exhaustive protease digestion of whole sperm, fluorometric anion-exchange high-performance liquid chromatography, and methylation analysis. The sulfate group was predominantly localized to the nonreducing terminus of the polySia chain. This is the first example of an alpha2,9-linked polySia structure in animal sperm. The polySia-containing sialoglycoprotein was present in sperm flagellum but not in the head. Furthermore, this sialoglycoprotein localized in the sperm lipid raft, which contains an enriched ganglioside (Neu5Acalpha2-->8Neu5Acalpha2-->6GlcCer), a receptor for sperm-activating peptide (speract), and its associated guanylate cyclase.

Animals↗