Shogo Oka

An ICAM-related neuronal glycoprotein, telencephalin, with brain segment-specific expression

Telencephalin (TLN) is a 130 kd glycoprotein expressed exclusively in neurons of the telencephalon, the most rostral brain segment. In the neurons, TLN is localized to soma-dendritic membrane but not to axonal membrane. In this study, we have cloned cDNA encoding rabbit and mouse TLN. The cDNA-derived primary structure of TLN predicts an integral membrane protein with nine tandem immunoglobulin-like domains in an extra-cellular region, a transmembrane domain, and a short cytoplasmic tail. The distal eight immunoglobulin-like domains of TLN show highest homology with the immunoglobulin-like domains of intracellular adhesion molecules (ICAMs) 1, 2, and 3/R. The structural similarity of TLN with ICAMs provides a new and strong link between immunoglobulin superfamily molecules in the nervous and immune systems. TLN is an example of a dendrite-associated cell adhesion molecule involved in the brains segmental organization, cell-cell interactions during dendritic development, and maintenance of functional neuronal networks.

Expression Cloning of a cDNA Encoding a Sulfotransferase Involved in the Biosynthesis of the HNK-1 Carbohydrate Epitope*

The HNK-1 carbohydrate epitope is expressed on several neural adhesion glycoproteins and as a glycolipid, and is involved in cell interactions. The structural element of the epitope common to glycoproteins and glycolipids has been determined to be sulfate-3-GlcAβ1→ 3Galβ1→4GlcNAc. The glucuronyltransferase and sulfotransferase are considered to be the key enzymes in the biosynthesis of this epitope because the rest of the structure occurs often in glycoconjugates. Here we describe the isolation of the rat sulfotransferase cDNA via an expression cloning strategy. The clone finally isolated predicts a protein of 356 amino acids, with characteristics of a type II transmembrane protein and with no sequence similarity to other known sulfotransferases. Both the enzyme expressed as a soluble fusion protein and homogenates of cells transfected with the full-length cDNA could transfer sulfate from a sulfate donor to acceptor substrates containing terminal glucuronic acid.

MOLECULAR CLONING AND FUNCTIONAL EXPRESSION OF A CDNA ENCODING A NEW MEMBER OF MIXED LINEAGE PROTEIN KINASE FROM HUMAN BRAIN

We have cloned a novel protein kinase from human cerebellum and named it LZK (leucine zipper-bearing kinase). The LZK cDNA encoded a 966-amino acid polypeptide that contains a kinase catalytic domain and double leucine/isoleucine zippers separated by a short spacer region. The amino acid sequence of the kinase catalytic domain was a hybrid between those in serine/threonine and tyrosine protein kinases, indicating that LZK belongs to the subfamily of the mixed lineage kinase (MLK) family. The kinase catalytic domain of LZK was most similar to DLK (Holtzman, L. B., Merritt, S.E., and Fan, G. (1994) J. Biol. Chem. 269, 30808–30817), MUK (Hirai, S., Izawa, M., Osada, S., Spyrou, G., and Ohno, S. (1996)Oncogene 12, 641–650), and ZPK (Reddy, U. R., and Presure, D. (1994) Biochem. Biophys. Res. Commun. 202, 613–620), which belong to the same subfamily of the MLK family. However, besides the kinase catalytic domain and double leucine/isoleucine zippers, there was no significant homology with known proteins. The recombinant LZK autophosphorylated in the presence of ATP and divalent cations, and exhibited serine/threonine kinase catalytic activity. Northern blot analysis revealed that LZK is expressed most strongly in the pancreas, with a pattern that differs from other MLKs. Expression of LZK in COS7 cells induced phosphorylation of c-Jun and activation of JNK-1, indicating the association of LZK in the c-Jun amino-terminal kinase/stress-activated protein kinase pathway. The expressed LZK was detected primarily in the membrane fraction, suggesting that LZK interacts with other cellular components in vivo.

Isolation and characterization of two distinct mannan-binding proteins from rat serum

Two binding proteins, which are specific for mannose and N-acetylglucosamine, were isolated from rat serum to homogeneity. The minor component [serum mannan-binding protein I (S-MBP-I)] was indistinguishable from rat liver mannan-binding protein (L-MBP). S-MBP-I had a molecular mass of about 200 kDa and consisted of about six identical 32-kDa subunits; the molecule had a collagen-like structure, and its properties were identical to those of L-MBP. S-MBP-I was also indistinguishable from L-MBP in immunochemical reactivity. Furthermore, the sequence of 15 NH2-terminal amino acids of S-MBP-I was identical to that of L-MBP, the complete primary structure of which has been elucidated [Drickamer, K., Dordal, M. S., and Reynolds, L. (1986) J. Biol. Chem. 261, 6878-6887; Oka, S., Itoh, N., Kawasaki, T., and Yamashina, I. (1987) J. Biochem. 101, 135-144]. The major component (S-MBP-II) had a molecular mass of about 650 kDa and consisted of about 20 identical 31-kDa subunits; it was immunochemically distinct from L-MBP and S-MBP-I, although the molecule had a collagen-like structure similar to L-MBP and S-MBP-I. Metabolic studies using [3H]leucine showed that S-MBP-II is a typical plasma protein turning over with a half-life of 1.6 days. S-MBP-I was unusual in its late appearance and rapid turnover rate in plasma. These results, together with the fact that L-MBP decayed with biphasic curves, suggest that a part of L-MBP is leaked from liver into plasma in the form of S-MBP-I.

Immunoglobulin superfamily molecules in the nervous system

Among the various types of membrane molecules involved in cell-cell interactions in the nervous system, we have focused in this review upon membrane proteins belonging to the immunoglobulin superfamily (IgSF). IgSF molecules are distinctive in that: (1) a large percentage of known neural adhesion molecules belongs to the IgSF; (2) they are homologous in structure (Ig domain), yet exhibit large variation of function in cell-cell interactions. The structure of IgSF molecules is briefly summarized in Section II, and each member of the IgSF which has been found in the nervous system is reviewed in Section III. In Section IV, we have discussed possible properties of yet-unknown nervous system IgSF molecules, on the assumption that nervous system IgSF molecules thus far discovered comprise only a small portion of those existing. Discussion is based upon an analogy with the immune system and upon knowledge of cell-cell interactions in the development of the nervous system. Our principal aims in this review are to summarize knowledge of neural IgSF molecules and to discuss the possibility that some IgSF molecules may encode in their structures instructions for recognizing, or for being recognized by, target neural cells. Further growth of knowledge of IgSF molecules may yield insights into the patterns of cell-cell interactions underlying the formation of neuronal circuits during development.

Structural Basis for Acceptor Substrate Recognition of a Human Glucuronyltransferase, GlcAT-P, an Enzyme Critical in the Biosynthesis of the Carbohydrate Epitope HNK-1

The HNK-1 carbohydrate epitope is found on many neural cell adhesion molecules. Its structure is characterized by a terminal sulfated glucuronyl acid. The glucuronyltransferases, GlcAT-P and GlcAT-S, are involved in the biosynthesis of the HNK-1 epitope, GlcAT-P as the major enzyme. We overexpressed and purified the recombinant human GlcAT-P from Escherichia coli. Analysis of its enzymatic activity showed that it catalyzed the transfer reaction for N-acetyllactosamine (Galβ1-4GlcNAc) but not lacto-N-biose (Galβ1-3GlcNAc) as an acceptor substrate. Subsequently, we determined the first x-ray crystal structures of human GlcAT-P, in the absence and presence of a donor substrate product UDP, catalytic Mn2+, and an acceptor substrate analogue N-acetyllactosamine (Galβ1-4GlcNAc) or an asparagine-linked biantennary nonasaccharide. The asymmetric unit contains two independent molecules. Each molecule is an α/β protein with two regions that constitute the donor and acceptor substrate binding sites. The UDP moiety of donor nucleotide sugar is recognized by conserved amino acid residues including a DXD motif (Asp195-Asp196-Asp197). Other conserved amino acid residues interact with the terminal galactose moiety of the acceptor substrate. In addition, Val320 and Asn321, which are located on the C-terminal long loop from a neighboring molecule, and Phe245 contribute to the interaction with GlcNAc moiety. These three residues play a key role in establishing the acceptor substrate specificity.

Purification and Characterization of a Glucuronyltransferase Involved in the Biosynthesis of the HNK-1 Epitope on Glycoproteins from Rat Brain

The glucuronyltransferase involved in the biosynthesis of the HNK-1 epitope on glycoproteins was purified to an apparent homogeneity from the Nonidet P-40 extract of 2-week postnatal rat forebrain by sequential chromatographies on CM-Sepharose CL-6B, UDP-GlcA-Sepharose 4B, asialo-orosomucoid-Sepharose 4B, Matrex gel Blue A, Mono Q, HiTrap chelating, and HiTrap heparin columns. The purified enzyme migrated as a 45-kDa protein upon SDS-polyacrylamide gel electrophoresis under reducing conditions, but eluted as a 90-kDa protein upon Superose gel filtration in the presence of Nonidet P-40, suggesting that the enzyme forms homodimers under non-denatured conditions. The enzyme transferred glucuronic acid to various glycoprotein acceptors bearing terminal N-acetyllactosamine structure such as asialo-orosomucoid, asialo-fetuin, and asialo-neural cell adhesion molecule, whereas little activity was detected to paragloboside, a precursor glycolipid of the HNK-1 epitope on glycolipids. These results suggested that the enzyme is specifically associated with the biosynthesis of the HNK-1 epitope on glycoproteins. Sphingomyelin was specifically required for expression of the enzyme activity. Stearoyl-sphingomyelin (18:0) was the most effective, followed by palmitoyl-sphingomyelin (16:0) and lignoceroyl-sphingomyelin (24:0). Interestingly, activity was demonstrated only for sphingomyelin with a saturated fatty acid,i.e. not for that with an unsaturated fatty acid, regardless of the length of the acyl group .

Characterization of recombinant human glucuronyltransferase I involved in the biosynthesis of the glycosaminoglycan-protein linkage region of proteoglycans.

We characterized the recombinant glucuronyltransferase I (GlcAT‐I) involved in the glycosaminoglycan‐protein linkage region biosynthesis. The enzyme showed strict specificity for Galβ1‐3Galβ1‐4Xyl, exhibiting negligible incorporation into other galactoside substrates including Galβ1‐3Galβ1‐O‐benzyl, Galβ1‐4GlcNAc and Galβ1‐4Glc. A comparison of the GlcAT‐I with another β1,3‐glucuronyltransferase involved in the HNK‐1 epitope biosynthesis revealed that the two β1,3‐glucuronyltransferases exhibited distinct and no overlapping acceptor substrate specificities in vitro. Nevertheless, the transfection of the GlcAT‐I cDNA into COS‐1 cells induced the significant expression of the HNK‐1 epitope. These results suggested that the high expression of the GlcAT‐I gene rendered the cells capable of synthesizing the HNK‐1 epitope.

Physical and Functional Association of Glucuronyltransferases and Sulfotransferase Involved in HNK-1 Biosynthesis

HNK-1 carbohydrate expressed predominantly in the nervous system is considered to be involved in cell migration, recognition, adhesion, and synaptic plasticity. Human natural killer-1 (HNK-1) carbohydrate has a unique structure consisting of a sulfated trisaccharide (HSO3-3GlcAβ1-3Galβ1-4GlcNAc-) and is sequentially biosynthesized by one of two glucuronyltransferases (GlcAT-P or GlcAT-S) and a sulfotransferase (HNK-1ST). Considering that almost all the HNK-1 carbohydrate structures so far determined in the nervous system are sulfated, we hypothesized that GlcAT-P or GlcAT-S functionally associates with HNK-1ST, which results in efficient sequential biosynthesis of HNK-1 carbohydrate. In this study, we demonstrated that both GlcAT-P and GlcAT-S were co-immunoprecipitated with HNK-1ST with a transient expression system in Chinese hamster ovary cells. Immunofluorescence staining revealed that these enzymes are mainly co-localized in the Golgi apparatus. To determine which domain is involved in this interaction, we prepared the C-terminal catalytic domains of GlcAT-P, GlcAT-S, and HNK-1ST, and we then performed pulldown assays with the purified enzymes. As a result, we obtained evidence that mutual catalytic domains of GlcAT-P or GlcAT-S and HNK-1ST are important and sufficient for formation of an enzyme complex. With an in vitro assay system, the activity of HNK-1ST increased about 2-fold in the presence of GlcAT-P or GlcAT-S compared with that in its absence. These results suggest that the function of this enzyme complex is relevant to the efficient sequential biosynthesis of the HNK-1 carbohydrate.

HNK-1 Glyco-epitope Regulates the Stability of the Glutamate Receptor Subunit GluR2 on the Neuronal Cell Surface

HNK-1 (human natural killer-1) glyco-epitope, a sulfated glucuronic acid attached to N-acetyllactosamine on the nonreducing termini of glycans, is highly expressed in the nervous system. Our previous report showed that mice lacking a glucuronyltransferase (GlcAT-P), a key enzyme for biosynthesis of the HNK-1 epitope, showed reduced long term potentiation at hippocampal CA1 synapses. In this study, we identified an α-amino-3-hydroxy-5-methylisoxazole propionate (AMPA)-type glutamate receptor subunit, GluR2, which directly contributes to excitatory synaptic transmission and synaptic plasticity, as a novel HNK-1 carrier molecule. We demonstrated that the HNK-1 epitope is specifically expressed on the N-linked glycan(s) on GluR2 among the glutamate receptors tested, and the glycan structure, including HNK-1 on GluR2, was determined using liquid chromatography-tandem mass spectrometry. As for the function of HNK-1 on GluR2, we found that the GluR2 not carrying HNK-1 was dramatically endocytosed and expressed less on the cell surface compared with GluR2 carrying HNK-1 in both cultured hippocampal neurons and heterologous cells. These results suggest that HNK-1 stabilizes GluR2 on neuronal surface membranes and regulates the number of surface AMPA receptors. Moreover, we showed that the expression of the HNK-1 epitope enhanced the interaction between GluR2 and N-cadherin, which has important roles in AMPA receptor trafficking. Our findings suggest that the HNK-1 epitope on GluR2 regulates cell surface stability of GluR2 by modulating the interaction with N-cadherin.