Yota Tatara

Identification of proteoglycan from salmon nasal cartilage.

There has been no structural information about the core protein of salmon nasal cartilage proteoglycan although its physiological activities have been investigated. Internal amino acid sequencing using nano-LC/MS/MS revealed that the salmon proteoglycan was aggrecan. Primer walk sequencing based on the amino acid information determined that the salmon aggrecan cDNA is comprised of 4207bp nucleotides predicted to encode 1324 amino acids with a molecular mass of 143,276. It exhibited significant similarities to predicted pufferfish aggrecan, zebrafish similar to aggrecan, zebrafish aggrecan, bovine aggrecan and human aggrecan isoform 2 precursor; whose amino acid identities were 56%, 55%, 49%, 31% and 30%, respectively. Salmon cartilage aggrecan had globular domains G1, G2 and G3 as in mammalian aggrecans. Neither the putative keratan sulfate attachment domain enriched with serine, glutamic acid and proline, nor the putative chondroitin sulfate attachment domain with repeating amino acid sequence containing serine-glycine, found in mammalian aggrecans were observed in salmon, however, random serine-glycine (or glycine-serine) sequences predicted to the sugar chain attachment sites were observed. Based on cDNA analysis and amino acid analysis after β-elimination, the ratio of serine attached to sugar chains was calculated to be approximately 37.7% of total serine, that is, 46 of 123 serine residues.

Biochemical and atomic force microscopic characterization of salmon nasal cartilage proteoglycan

Biological activities of salmon nasal cartilage proteoglycan fractions are known, however, structural information is lacking. Recently, the major proteoglycan of this cartilage was identified as aggrecan. In this study, global molecular images and glycosaminoglycan structure of salmon nasal cartilage aggrecan purified from 4M guanidine hydrochloride extract were analyzed using HPLCs and atomic force microscopy with bovine tracheal cartilage aggrecan as a control. The estimated numbers of sulfates per disaccharide unit of chondroitin sulfate chains of salmon and bovine aggrecans were similar (approximately 0.85). However, the disaccharide composition showed a higher proportion of chondroitin 6-sulfate units in salmon aggrecan, 60%, compared to 40% in bovine. Gel filtration HPLC and monosaccharide analysis showed the salmon aggrecan had a lower number (approximately one-third), but 1.5-3.3 times longer chondroitin sulfate chains than the bovine aggrecan. Atomic force microscopic molecular images of aggrecan supported the images predicted by biochemical analyses.

AMBRA1, a novel α-synuclein-binding protein, is implicated in the pathogenesis of multiple system atrophy

The accumulation of abnormal α‐synuclein is the major histopathological feature of Lewy body disease and multiple system atrophy (MSA), which are referred to as synucleinopathies. Cytoplasmic degradation systems, such as the autophagy‐lysosome and proteasome pathways, are involved in their pathogenesis. Autophagy is tightly regulated by several upstream proteins including UNC‐51‐like kinase 1/2, beclin1, vacuolar protein sorting‐associated protein 34 and autophagy/beclin1 regulator 1 (AMBRA1). Recently, we revealed that both cortical and brainstem‐type Lewy bodies were immunopositive for several upstream proteins of autophagy. Therefore, we conducted the present study to elucidate the role of upstream proteins of autophagy in the pathogenesis of MSA. Pathological and biochemical analyses using human brain samples revealed that AMBRA1 is a component of the pathological hallmarks of MSA and upstream proteins of autophagy are impaired in the MSA brain. In vitro and in vivo analyses revealed a ninefold stronger affinity of AMBRA1 with α‐synuclein phosphorylated at serine 129 compared with non‐phosphorylated α‐synuclein. Furthermore, a weak but significant correlation between AMBRA1 overexpression and reduction of abnormal α‐synuclein was observed. Silencing AMBRA1 function caused aggregates of α‐synuclein in the cytoplasm of mouse primary cultured neurons, which was simulated by the treatment of Bafilomycin, an autophagy inhibitor. Our results demonstrated for the first time that AMBRA1 is a novel hub binding protein of α‐synuclein and plays a central role in the pathogenesis of MSA through the degradative dynamics of α‐synuclein. These results raise the possibility that molecular modulation targeting AMBRA1 can be a promising candidate for the treatment of synucleinopathies.

Epiphycan from salmon nasal cartilage is a novel type of large leucine-rich proteoglycan

Chum salmon (Oncorhynchus keta) nasal cartilage was examined by next-generation DNA sequencing and mass spectrometric analyses, and 14 types of proteoglycans including epiphycan (EPY) were found. A cDNA encoding EPY was cloned and sequenced. The cDNA encoded 589 amino acids comprised a glycosaminoglycan (GAG) domain containing 55 potential GAG-modified sites (Ser-Gly and/or Gly-Ser), a cysteine cluster and 6 leucine-rich repeats. EPY was purified from salmon nasal cartilage and the structure of the GAG was characterized. As a result of unsaturated disaccharide analysis, GAG was found to be composed of chondroitin 6-sulfate (58.0%), chondroitin 4-sulfate (26.5%) and non-sulfated chondroitin (15.3%). The average molecular weight of GAG was estimated to be 3.0 × 10(4). Ser-100 and Ser-103 were identified as serine residues substituted by GAG chains by chemical modification and mass spectrometric analysis. More than 50 serine residues were assumed to be substituted by GAG chains. EPY is heavily substituted by chondroitin sulfate, giving an overall molecular weight of just under 2 × 10(6). EPY from salmon nasal cartilage is a novel type of large leucine-rich proteoglycan.

Chondroitin sulfate cluster of epiphycan from salmon nasal cartilage defines binding specificity to collagens

Epiphycan (EPY) from salmon nasal cartilage has a glycosaminoglycan (GAG) domain that is heavily modified by chondroitin 4-sulfate and chondroitin 6-sulfate. The functional role of the GAG domain has not been investigated. The interaction of EPY with collagen was examined in vitro using surface plasmon resonance analysis. EPY was found to bind to type I collagen via clustered chondroitin sulfate (CS), while a single chain of CS was unable to bind. Types I, III, VII, VIII and X collagen showed high binding affinity with EPY, whereas types II, IV, V, VI and IX showed low binding affinities. Chemical modification of lysine residues in collagen decreased the affinity with the clustered CS. These results suggest that lysine residues of collagen are involved in the interaction with the clustered CS, and the difference in lysine modification defines the binding affinity to EPY. The clustered CS was also involved in an inter-saccharide interaction, and formed self-associated EPY. CS of EPY promoted fibril formation of type I collagen.

Hyaluronan-chondroitin hybrid oligosaccharides as new life science research tools.

Hyaluronan and chondroitin are glycosaminoglycans well-known as components of pharmaceutical agents and health foods. From these attractive molecules, using transglycosylation reaction of testicular hyaluronidase, we synthesized hybrid neo-oligosaccharides not found in nature. We also found a new site between the chondroitin disaccharide unit and hyaluronan disaccharide unit recognized by a hyaluronan lyase specific to hyaluronan using these hybrid oligosaccharides as substrates. We hope that these hybrid oligosaccharides will help to elucidate the involvement of hyaluronan, chondroitin, and chondroitin sulfates in the mechanisms of cell functions and diseases, based on the structures of these glycosaminoglycans.

Novel roles of glycosaminoglycans in the degradation of type I collagen by cathepsin K

Glycosaminoglycans (GAGs) and collagen are the major organic components of bone matrix. However, their roles and functional relationships remain elusive. To investigate the role of GAGs in bone matrix degradation, the effects of GAGs on collagen were examined under acidic conditions that recapitulate the microenvironment of osteoclast resorption pits. We found that sulfated GAGs protect collagen fibrils against acid denaturation. Scanning electron microscopy demonstrated that collagen fibrils retain the fibril structure at pH 4.0 in the presence of chondroitin 6-sulfate. By surface plasmon resonance analysis, we found that sulfated GAGs, but not non-sulfated GAGs, bind to triple-helix type I collagen below pH 4.5. The binding of collagen in an acidic solution was dependent upon the GAG sugar chain length. Functionally, the acid-resistant collagen fibrils generated in the presence of sulfated GAGs were resistant to cathepsin K degradation in vitro below pH 4.0. As the pH increased from 4.0 to 5.0, the acid-resistant collagen fibrils were degraded by cathepsin K. Our results highlight the possibility that the interaction between GAGs and collagen under acidic conditions has a regulatory impact on cathepsin K-mediated bone degradation.

Identification of Pin1-Binding Phosphorylated Proteins in the Mouse Brain

To determine the role of Pin1 in the neurotransmission pathway, Pin1-binding proteins in mouse brain extract were identified. The Pin1-binding proteins were extracted from mouse brain homogenate, and the trypsin-digested peptides were analyzed by nano-liquid chromatography tandem mass spectrometry (LC-MS/MS). Proteins that involve the neurotransmission pathway, such as synapsin I, synapsin II, and calcium/calmodulin-dependent protein kinase type II (CaMKII), were identified in a Mascot search. Pull-down and immunoprecipitation assay indicated that Pin1 binds CaMKII in a phosphorylation-specific manner. It was assumed that Pin1 participates in the neurotransmission pathway involving the phosphorylation signal by CaMKII.

Synthesis of N-acetyl Glucosamine Analogs as Inhibitors for Hyaluronan Biosynthesis

Elevated hyaluronan expression is a hallmark of many types of cancer. Therefore, inhibition of hyaluronan biosynthesis can potentially slow the growth of tumor cells. Herein, we explore a chain termination strategy to reduce hyaluronan synthesis by tumor cells. Several analogs of glucosamine were prepared, which contained modifications at the C-3 positions. These analogs can possibly cap the nonreducing end of a growing hyaluronan chain, thus lowering the amount of hyaluronan synthesized. Upon incubation with pancreatic cancer cells, a fluorine-containing glucosamine analog was found to exhibit significant inhibitory activities of hyaluronan synthesis. Furthermore, it drastically reduced the proliferation of cancer cells. Supplemental materials are available for this article. Go to the publishers online edition of Journal of Carbohydrate Chemistry to view the supplemental file.

A mechanism for evasion of CTL immunity by altered O-glycosylation of HLA class I

Anti-tumour immunity by cytotoxic T-lymphocytes (CTLs) is essential to suppress tumour progression. Cancer cells that evade CTL immunity proliferate in the host, promoting metastasis, but mechanisms underlying this capacity remain unknown. Here we report that bladder cancer cells metastasized to lymph nodes evade CTL immunity by a new mechanism via altered glycosylation. CTLs normally recognize and kill cancer cells presenting antigenic peptides on human leukocyte antigen (HLA) class I. We show bladder cancer cells expressing the O-glycan processing enzyme, core2 β-1,6-N-acetylglucosaminyltransferase (C2GnT) exhibit HLA class I O-glycan modified with poly-N-acetyllactosamine and are highly susceptible to CTL. In those cells, poly-N-acetyllactosamine on HLA class I O-glycan binds galectin-3 to form a cell-surface molecular lattice, enabling efficient cell-surface retention of HLA class I. In contrast, bladder cancer cells in which C2GnT is downregulated show decreased levels of poly-N-acetyllactosamine on HLA class I O-glycans, attenuating lattice formation and reducing the cell-surface half-life of HLA class I. These tumour cells present antigenic peptides less efficiently, thereby evading CTL lysis and facilitating metastasis.