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N-Acetylglucosamine 6-O-sulfotransferases (GlcNAc6STs) catalyze the transfer of sulfate from 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to the C-6 position of non-reducing N-acetylglucosamine. N-acetylglucosamine 6-O-sulfotransferase-1 (GlcNAc6ST-1) is the first cloned GlcNAc6ST and is involved in the synthesis of the l-selectin ligand. We noticed conserved C-terminal segments among GlcNAc6STs and produced mutant enzymes to reveal the functional significance. Mutant enzymes were transiently expressed as fusion proteins with protein A in COS-7 cells, and some of them were purified to homogeneity by IgG Sepharose column chromatography. Deletion of a C-terminal segment (amino acid numbers 479–483) resulted in a complete loss of the activity, when assayed using GlcNAcβ1-6ManOMe as a substrate. Upon site-directed mutagenesis of the C-terminal region, three mutants, L477A, L478A and L483A, exhibited reduced activity. The KM values for GlcNAcβ1-6ManOMe of L477A and L478A were 4 times higher than the KM of the wild-type enzyme, while that of L483A was unchanged. On the other hand the KM for PAPS of L483A was 3 times higher than that of the wild-type enzyme, while the values of L477A and L478A were unchanged. Furthermore, the L477A mutant acted on a core 3 structure (GlcNAcβ1-3GalNAc-pNP), while the wild-type enzyme does not. These results demonstrate a role for leucine residues in the C-terminal region in the enzymatic activity. [ABSTRACT FROM PUBLISHER]

N-Acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST) catalyzes the transfer of sulfate from 3′-phosphoadenosine 5′-phosphosulfate to the C-6 position of non-reducing GlcNAc. Human GlcNAc6ST-1 was expressed as a fusion protein with protein A in an insect cell line (Tn 5 cells) using the baculovirus system. The recombinant enzyme was purified to homogeneity by IgG Sepharose column chromatography. The substrate specificity and the kinetic properties of the enzyme were similar to those of the enzyme expressed in the mammalian system. The purified recombinant enzyme was used to synthesize 6-sulfo GlcNAcβ1–3Galβ1–4Glc, which was identified by time of flight mass spectrometry. This sulfated trisaccharide served as a better substrate for microsomal galactosyltransferase from the mouse colon compared to 6-sulfo GlcNAc. The purified recombinant enzyme was also used to sulfate oligosaccharide chains on fibrinogen after enzymatic desialylation and degalactosylation to expose nonreducing GlcNAc residues. It is known that desialylation greatly increases the rate of clotting of fibrinogen after the addition of thrombin. Subsequent sulfation of desialylated and degalactosylated fibrinogen slightly decreased the rate of clotting. The recombinant GlcNAc6ST-1 is a useful reagent for 6-sulfate exposed GlcNAc residues both in oligosaccharides and in glycoproteins. [ABSTRACT FROM PUBLISHER]

Endo-β-N-acetylglucosaminidase D (Endo D) produced by Streptococcus pneumoniaecleaves the di-N-acetylchitobiose structure in asparagine-linked oligosaccharides. The enzyme generally acts on complex type oligosaccharides after removal of external sugars by neuraminidase, β-galactosidase, an β-N-acetylglucosaminidase. We cloned the gene encoding the enzyme and expressed it as a periplasm enzyme in Escherichia coil The first 37 amino acids in the predicted sequence are removed in the mature enzyme, yielding a protein with a molecular mass of 178 kDa. The substrate specificity of the recombinant enzyme is indistinguishable from the enzyme produced by S. pneumoniae. Endo-β-N-acetylglucosaminidase A (Endo A) from Arthrobacter protophormiae, the molecular mass of which is 72 kDa, had 32% sequence identity to Endo D, starting from the N-terminal sides of both enzymes, although Endo A hydrolyzes high-mannose type oligosaccharides and does not hydrolyze complex type ones. Endo D is not related to endo-β-N-acetylglucosaminidases H, F1, F2, or F3, which share common structural motifs. Therefore, there are two distinct groups of endo-β-n-acetylglucosaminidases acting on asparagine-linked oligosaccharides. The C-terminal region of Endo D shows homology to β-galactosidase and β-N-acetylglucosaminidase from S. pneumoniae and has an LPXTG motif typical of surface-associated proteins of Gram-positive bacteria. It is possible that Endo D is located on the surface of the bacterium and, together with other glycosidases, is involved in virulence. [ABSTRACT FROM AUTHOR]

Prp8 is a splicing factor of 220 kDa originally identified in yeast and is a component of the U5 small nuclear ribonucleoprotein particle. Mouse Prp8 cDNA was cloned and shown to share 62.6 and 68.2% sequence identity with the yeast homologue at the amino acid and nucleotide level, respectively, while it differs by only 3 amino acid residues from the human homologue. During mouse embryogenesis, Prp8 is expressed intensely at day 9.5 of gestation, and its expression decreases progressively during embryogenesis. In adult mice, Prp8 is expressed strongly in the testis and moderately in the ovary. In situ hybridization analysis revealed that Prp8 is preferentially expressed in the outer cell layer in the testis, probably in the spermatogonia and primary spermatocytes, and in granulosa cells in the ovary. In Caenorhabditis elegans, microinjection of a double stranded RNA corresponding to a portion of the Prp8 sequence results in the arrest of embryogenesis at the late-gastrulation stage. These results suggest that Prp8 plays an important role in reproduction and development. Prp8 was shown to bind to midkine (MK), a heparin-binding growth factor. Since Prp8 expression partially overlaps with the sites of action of MK, it is possible that binding to Prp8 is involved in part of MK signaling. [ABSTRACT FROM AUTHOR]

MGC-24/CD164 is a sialomucin expressed in many normal and cancerous tissues. In humans, soluble and transmembrane forms of MGC-24 are produced by alternative splicing. The total MGC-24 RNA level was found to be lower in human colorectal carcinomas as compared with the adjacent normal mucosal tissues. Lower MGC-24 mRNA levels in colon carcinomas and in the adjacent normal mucosa epithelium correlate with lymphatic vessel invasion by the carcinoma. The ratio of the soluble form to the transmembrane form of the mRNA in colorectal carcinomas was determined by ribonuclease protection assay. Higher ratios were correlated with less venous invasion and less remote metastasis, which became evident during postoperative observation. [ABSTRACT FROM AUTHOR]

Midkine (MK) is a heparin-binding growth factor involved in diverse biological phenomena, e.g. neuronal survival, carcinogenesis, and tissue repair. ME expression is detected mainly in the kidney in adult mice. In this study, we show that, at a dose that can induce recoverable renal damage and induce apoptosis, cisplatin (CDDP) transiently suppressed MX expression in mouse kidney. In vitro, CDDP suppressed MX expression and induced apoptosis in cultured G401 cells, a Wilms, tumor cell line. Exogenous MX protein partially rescued (3401 cells from CDDP-induced apoptosis. ME enhanced the expression of Bcl-2, but not that of Bcl-x1 in 0401 cells in a dose-dependent manner, and it prevented the Bcl-2 reduction due to CDDP. Moreover, Bcl-2 expression in mouse kidney was also transiently suppressed by CDDP treatment, the expression proffle being similar to that of ME. These results imply that ME exerts cytoprotective activity toward a damaging insult, presumably at least in part through enhancement of the expression of Bcl-2 [ABSTRACT FROM AUTHOR]

Midkine (MK) is a heparin-binding growth/differentiation factor implicated in the control of development and repair of various tissues. To investigate the roles of MK in embryogenesis and in regulation of wound healing, we utilized a system of collagen gel contraction by dermal fibroblasts, which provides an in vitro model for studying fibro-blast-collagen interactions important in various physiological and pathological phe-nomena. MK enhanced gel contraction 8–24 h after plating, and its effect was inhibited by anti-MK antibody. The effect was reduced after 48 h, while TGF-p continued to be active in the later stage. Morphologically, MK-treated fibroblasts tended to be elongated more frequently than control fibroblasts. The effect of MK in the early stage of gel contraction suggests a role of MK as a modulator of cell-matrix interactions. [ABSTRACT FROM AUTHOR]

Zep, a novel 49 kDa zinc finger protein, was found in the brain of day-13 mouse embryos and cloned. Zep contains two C2H2-type zinc finger motifs close to the N-terminal region. The majority of the molecule is composed of a proline-rich domain showing similarity to proline-rich domains in transcription factors and a salivary proline-rich protein. In addition to the proline-rich domain, Zep has an acidic domain and a serine/threonine-rich domain, all of which are frequently found in many transcription factors. The overall organization of Zep shows no similarity to any other proteins. There is a nuclear localization signal in Zep, and the Zep-GFP (green fluorescent protein) fusion protein is located predominantly in the nucleus. In the day-13 mouse embryo, Zep is strongly expressed in the nervous system, i.e. brain, spinal cord, and dorsal root ganglia, with strong to weak expression observed in other regions. Zep continues to be strongly expressed in the neonatal brain; however, its expression is weak in the brain and spinal cord of adult mice. In situ hybridization reveals strong signals for Zep mRNA in the cerebellum and olfactory bulb with moderate signals detected in the hippocampus and cortex. Strong Zep expression is observed in adult thymus, lung, spleen, testis, and ovary. Zep may be involved in the formation and remodeling of various tissues including nervous tissue, probably through transcriptional regulation. [ABSTRACT FROM AUTHOR]

N-Acetylglucosamine-6-O-sulfotransferase catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 6 of a non-reducing N-acetylglucosamine (GlcNAc) residue. We have cloned human GlcNAc-6-O-sulfotransferase cDNA, based on the sequence homology to cloned cDNA of mouse GlcNAc-6-O-sulfotransferase. The predicted protein sequence of the human enzyme was highly homologous to that of the mouse enzyme; in the 363 amino acid stretch of the catalytic region, the two proteins were nearly identical except for conservative changes in 3 amino acid residues. The expressed enzyme transferred sulfate to GlcNAcβl-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc. Co-transfection of the enzyme cDNA and fucosyltransferase VII cDNA into COS-7 cells resulted in cell surface expression of 6-sulfo sialyl Lewis X. Fluorescence in situ hybridization analysis revealed that the GlcNAc-6-O-sulfotransferase gene is located on human chromosome 7q31. mRNA of the human enzyme was strongly expressed in the bone marrow, peripheral blood leukocytes, spleen, brain, spinal cord, ovary, and placenta, and moderate levels of expression were observed in many organs including lymph nodes and thymus. In situ hybridization with the mouse system showed that the transcript was localized in specific regions of the brain, i.e. pyramidal cells in the CA3 subregion of the hippocampus, cerebellar nucleus and Purkinje cells. Among human tumor cells, strong expression of the mRNA was found in MOLT-4 and Jarkat lymphoblastic leukemia cells, Raji lymphoma cells, K-562 chronic myelogeneous leukemia cells, U251 glioma cells, and G361 melanoma cells. Carbohydrate structures synthesized by the sulfotransferase may be involved in various aspects of the differentiation and behavior of blood cells, their progenitor cells, and neurons in the central nervous system. [ABSTRACT FROM AUTHOR]

The removal of N-terminally located clusters of basic amino acids (N-tail) or C-terminally located clusters of basic amino acids (C-tail) from the midkine (MK) molecule severely reduced its neurite-promoting activity. However, experiments involving chemically synthesized MK derivatives revealed that the roles of the N-tail and C-tail were mostly indirect ones, i.e. they probably maintain the steric arrangements of the N-terminal and C-terminal halves. In particular, the C-domain, which is the C-terminal half devoid of the C-tail, retained considerable neurite-promoting activity when it was uniformly coated on a dish. The removal of the N-tail or C-tail also reduced the enhancing activity of plasminogen activator (PA) in aortic endothelial cells, although the effect was lower. There are two heparin-binding sites in the C-domain, Clusters I and II. A mutation in Cluster I [R78↑Q] affected the PA-enhancing activity only slightly, and a mutation in Cluster II [K83K74↑QQ] abolished the activity, while both mutations are known to reduce the neurite-promoting activity moderately. Therefore, the two heparin-binding sites in the C-domain play different roles in these two activities. Indeed, heparin exhibited different effects on these two activities. We also observed that intact MK was required for ordered neurite-promotion along the path of MK; one possible interpretation of this is that the N-terminal half is necessary for the stability of the molecule. Furthermore, K76 and K99 were found to be required for the secretion of MK; i.e. mutants in which one of these K residues was changed to Q were produced in the host cells, but not found in the medium. [ABSTRACT FROM AUTHOR]

The heparin-binding growth factor midkine (MK) has been implicated in neuron growth, angiogenesis, and inflammation. In this study, to elucidate the involvement of MK in the development of pathologies associated with uremia, we examined the serum MK levels in patients receiving hemodialysis (HD) by a highly sensitive enzyme-linked immunoassay. Although no significant difference was found between control serum and serum before dialysis in HD patients, serum MK levels increased significantly at the early stage of HD sessions using heparin and gradually decreased after dialysis. In normal controls, intravenous administration of heparin induced a similar sudden increase of MK, but the subsequent decrease was also rapid. In an in vitro study, MK was released in time- and heparin-dose dependent manner from cultured vessels, but not from peripheral leukocytes. These results indicate that, in HD patients, MK is released mainly from endothelial cells immediately after administration of heparin during HD and disappears gradually from blood due to renal impairment. This phenomenon might affect some complications associated with HD. [ABSTRACT FROM AUTHOR]

Midkine (MK) is a heparin-binding growth factor that has been implicated in neural survival and differentiation, fibrinolysis, and carcinogenesis. It is expressed in the nervous system during early Xenopus development. In the present study, we demonstrated that injection of vegetal blastomeres with Xenopus MK at the 8-cell stage results in incomplete invagination. In the case of dorsal vegetal injection, hypertrophic neural tissue is produced. Animal caps isolated from embryos that have been injected with Xenopus MK and cultured with activin do not elongate, and all mesoderm markers examined, including both head and trunk/tail ones, are greatly diminished. In contrast, head-specific neural markers, XANF-1 and Xotx2, are induced, while trunk/tail neural markers, XlHbox6 and F-spondin, are decreased. Moreover, MK showes the same effects in animal caps injected with Xenopus Smad2 mRNA. [ABSTRACT FROM AUTHOR]

Midkine (MK) is a retinoic acid-inducible heparin-binding cytokine. In the inflammatory synovitis of rheumatoid arthritis and osteoarthritis, MK was detected in synovial fluid, synoviocytes, and endothelial cells of new blood vessels. Normal synovial fluid and noninflammatory synovial tissue did not contain detectable MK. Therefore, MK showed inflammation-associated expression in these cases. Furthermore, MK was found to promote chemotaxis of neutrophils in the range of 10 ng/ml. The mode of action of MK was found to be haptotactic; the substrate-bound form of MK was the active one. MK is also known to promote fibrinolysis. These activities of MK are in agreement with the modes of MK expression in various pathological statuses, and thus MK is proposed to be an important molecule regulating inflammatory responses. [ABSTRACT FROM AUTHOR]

Midkine (MK) is a growth factor that promotes neurite outgrowth and survival of neurons, and enhances the plasminogen activator in endothelial cells. A highly sensitive enzyme-linked immunoassay for MK was developed, involving affinity-purified anti-MK antibodies, their biotinylated form, and avidin-β-galactosidase. The amount of bound avidin-β-galactosidase was determined using a fluorogenic substrate, 4-methylumbelliferyl-β-D-galactoside. This method allowed the detection of human and mouse MK in the range of 50 pg-10 ng. Pleiotrophin, which is related to MK in its amino acid sequence, did not show any cross reactivity. Employing this method, the MK levels in the developing mouse brain were determined. The MK level was 2 μg/g of wet tissue on the 12th day of gestation, and then steadily decreased during embryogenesis and postnatal development to 30 ng/g two months after birth. The assay method can also be applied to serum samples. Although the MK levels in the sera of normal human subjects were low or undetectable, 0.6-8 ng/ml of MK was detected in samples in the majority of cases of hepatocellular carcinomas. [ABSTRACT FROM AUTHOR]

By means of a baculovirus expression system, a large amount of mouse midkine (MK) was produced. The protein was purified to homogeneity by heparin-Sepharose column chro-matography. The purified protein was of a mature type; the signal peptide was cleaved at the expected site. To examine the neurite-guiding activity of MK, rat embryonic brain cells (embryonic days 17–18) were cultured on plates coated with purified MK in a grid pattern. The cells attached to and extended their neurites along the substrate pattern. This interaction was strongly inhibited by heparin, but not by other glycosaminoglycans. Treatment of the cells with heparitinase was effective for inhibiting their adhesion to the substrate. These data suggest that the heparin-like domain on cell surface heparan sulfate proteoglycan is the primary site for MK binding upon interaction with nerve cells. [ABSTRACT FROM AUTHOR]