Reiko Mineki

TANDEM REPEAT STRUCTURE OF RHAMNOSE-BINDING LECTIN FROM CATFISH (SILURUS ASOTUS) EGGS

The primary structure of catfish (Silurus asotus) egg lectin (SAL) was determined. SAL cDNA contained 1448-bp nucleotides and 308 amino acid residues, deduced from open reading frame. The SAL mature protein composed of 285-amino acid residues was followed by a predicted signal sequence having 23 residues. The mRNA of SAL was found to be expressed in eggs, but not in liver. SAL is composed of three tandem repeat domain structures divided into exactly 95 amino acid residues each, and all cysteine positions of each domain were completely conserved. Sequence homologies between the three domains, termed D1 (1-95), D2 (96-190) and D3 (191-285), were as follows; D1-D2, 28%; D2-D3, 33%; D1-D3, 43%. Two conserved peptide motifs, -(AN)YGR(TD)S(T)XCS(TGR)P- and -DPCX(G)T(Y)KY(L)-, appear to exist at the N- and C-terminal regions of each domain, respectively. The kinetic parameters of SAL obtained by measuring surface plasmon resonance were as follows: K(a) (M(-1)) for neohesperidosyl-BSA, 7. 1 x 10(6); for melibiosyl-BSA, 4.9 x 10(6); and for lactosyl-BSA, 5. 2 x 10(5). These results show that RBLs including SAL comprise a family of alpha-galactosyl binding lectins having characteristic tandem repeat domain structures.

Multiple Molecular Interactions Implicate the Connectin/Titin N2A Region as a Modulating Scaffold for p94/Calpain 3 Activity in Skeletal Muscle

p94/calpain 3 is a skeletal muscle-specific Ca2+-regulated cysteine protease (calpain), and genetic loss of p94 protease activity causes muscular dystrophy (calpainopathy). In addition, a small in-frame deletion in the N2A region of connectin/titin that impairs p94-connectin interaction causes a severe muscular dystrophy (mdm) in mice. Since p94 via its interaction with the N2A and M-line regions of connectin becomes part of the connectin filament system that serves as a molecular scaffold for the myofibril, it has been proposed that structural and functional integrity of the p94-connectin complex is essential for health and maintenance of myocytes. In this study, we have surveyed the interactions made by p94 and connectin N2A inside COS7 cells. This revealed that p94 binds to connectin at multiple sites, including newly identified loci in the N2A and PEVK regions of connectin. Functionally, p94-N2A interactions suppress p94 autolysis and protected connectin from proteolysis. The connectin N2A region also contains a binding site for the muscle ankyrin repeat proteins (MARPs), a protein family involved in the cellular stress responses. MARP2/Ankrd2 competed with p94 for binding to connectin and was also proteolyzed by p94. Intriguingly, a connectin N2A fragment with the mdm deletion possessed enhanced resistance to proteases, including p94, and its interaction with MARPs was weakened. Our data support a model in which MARP2-p94 signaling converges within the N2A connectin segment and the mdm deletion disrupts their coordination. These results also implicate the dynamic nature of connectin molecule as a regulatory scaffold of p94 functions.

Characterization of Cln3p, the gene product responsible for juvenile neuronal ceroid lipofuscinosis, as a lysosomal integral membrane glycoprotein

Juvenile neuronal ceroid lipofuscinosis (JNCL) is an autosomal recessively inherited lysosomal storage disease involving a mutation in the CLN3 gene. The sequence of CLN3 was determined in 1995; however, the localization of the CLN3 gene product (Cln3p) was not confirmed. In this study, we investigated endogenous Cln3p using two peptide antibodies raised against two distinct epitopes of murine Cln3p. Identification of the liver 60 kDa protein as Cln3p was ascertained by amino acid sequence analysis using tandem mass spectrometry. Liver Cln3p was predominantly localized in the lysosomal membranes, not in endoplasmic reticulum (ER) or Golgi apparatus. As the tissue concentration of brain Cln3p was much lower than that of liver Cln3p, it could be detected only after purification from brain extract using anti‐Cln3p IgG Sepharose. The apparent molecular masses of liver Cln3p and brain Cln3p were determined to be about 60 kDa and 55 kDa, respectively. Both brain and liver Cln3p were deglycosylated by PNGase F treatment to form polypeptides with almost the same molecular mass (45 kDa). However, they were not affected by Endo h treatment. In addition, it was also elucidated that the amino terminal region of Cln3p faces the cytosol.

Maturation of the activities of recombinant mite allergens Der p 1 and Der f 1, and its implication in the blockade of proteolytic activity

Recombinant pro‐Der p 1 expressed in yeast Pichia pastoris was convertible into the prosequence‐removed mature Der p 1 with full activities of cysteine protease and IgE‐binding with or without N‐glycosylation of the mature sequence as well as pro‐Der f 1. The active recombinant variants will be the basis for various future studies. The major N‐terminus of pro‐Der p 1 with low proteolytic activity was the putative signal‐cleavage site, while that of pro‐Der f 1 contained not only the equivalent site but also 21 residues downstream, and pro‐Der f 1 retained significant activity. Contribution of the N‐terminal region of the Der p 1 prosequence including an N‐glycosylation motif on effective inhibition of proteolytic activity of pro‐Der p 1 was suggested.

Autolysosomal membrane-associated betaine homocysteine methyltransferase. Limited degradation fragment of a sequestered cytosolic enzyme monitoring autophagy.

We compared the membrane proteins of autolysosomes isolated from leupeptin-administered rat liver with those of lysosomes. In addition to many polypeptides common to the two membranes, the autolysosomal membranes were found to be more enriched in endoplasmic reticulum lumenal proteins (protein-disulfide isomerase, calreticulin, ER60, BiP) and endosome/Golgi markers (cation-independent mannose 6-phosphate receptor, transferrin receptor, Golgi 58-kDa protein) than lysosomal membranes. The autolysosomal membrane proteins include three polypeptides (44, 35, and 32 kDa) whose amino-terminal sequences have not yet been reported. Combining immunoblotting and reverse transcriptase-polymerase chain reaction analyses, we identified the 44-kDa peptide as the intact subunit of betaine homocysteine methyltransferase and the 35- and 32-kDa peptides as two proteolytic fragments. Pronase digestion of autolysosomes revealed that the 44-kDa and 32-kDa peptides are present in the lumen, whereas the 35-kDa peptide is not. In primary hepatocyte cultures, the starvation-induced accumulation of the 32-kDa peptide occurs in the presence of E64d, showing that the 32-kDa peptide is formed from the sequestered 44-kDa peptide during autophagy. The accumulation is induced by rapamycin but completely inhibited by wortmannin, 3-methyladenine, and bafilomycin. Thus, detection of the 32-kDa peptide by immunoblotting can be used as a streamlined assay for monitoring autophagy.

Novel Mitochondrial Complex II Isolated from Trypanosoma cruzi Is Composed of 12 Peptides Including a Heterodimeric Ip Subunit

Mitochondrial respiratory enzymes play a central role in energy production in aerobic organisms. They differentiated from the α-proteobacteria-derived ancestors by adding noncatalytic subunits. An exception is Complex II (succinate: ubiquinone reductase), which is composed of four α-proteobacteria-derived catalytic subunits (SDH1-SDH4). Complex II often plays a pivotal role in adaptation of parasites in host organisms and would be a potential target for new drugs. We purified Complex II from the parasitic protist Trypanosoma cruzi and obtained the unexpected result that it consists of six hydrophilic (SDH1, SDH2N, SDH2C, and SDH5-SDH7) and six hydrophobic (SDH3, SDH4, and SDH8-SDH11) nucleus-encoded subunits. Orthologous genes for each subunit were identified in Trypanosoma brucei and Leishmania major. Notably, the iron-sulfur subunit was heterodimeric; SDH2N and SDH2C contain the plant-type ferredoxin domain in the N-terminal half and the bacterial ferredoxin domain in the C-terminal half, respectively. Catalytic subunits (SDH1, SDH2N plus SDH2C, SDH3, and SDH4) contain all key residues for binding of dicarboxylates and quinones, but the enzyme showed the lower affinity for both substrates and inhibitors than mammalian enzymes. In addition, the enzyme binds protoheme IX, but SDH3 lacks a ligand histidine. These unusual features are unique in the Trypanosomatida and make their Complex II a target for new chemotherapeutic agents.

Analysis of potential diagnostic biomarkers in cerebrospinal fluid of idiopathic normal pressure hydrocephalus by proteomics

SummaryBackground. The pathogenesis of idiopathic normal pressure hydrocephalus (INPH) is unknown, and the syndrome of INPH remains a diagnostic and therapeutic challenge. The present study investigated the disease-specific proteins that aid in the diagnosis and treatment of INPH and thus to study their role in the disease process.Methods. A comparative proteomic analysis was used for clinical screening of cerebrospinal fluid (CSF) proteins in 15 patients with INPH and compared with 12 normal subjects. Furthermore, enzyme linked immunosorbent assay (ELISA) was performed for comparison with CSF proteins between individual INPH patients and controls.Results. Seven proteins and their isoforms, including leucine-rich α-2-glycoprotein (LRG), α1-antichymotrypsin, apolipoprotein D, apolipoprotein J, haptoglobin α1, serum albumin, and α-1-microglobulin/bikunin precursor showed significant changes in CSF of INPH patients compared with controls by proteomic analysis. And significant higher CSF levels of LRG in INPH patients compared with controls were found by ELISA.Conclusions. These results indicate that there are significant differences in the expression of certain proteins in the CSF of patients with INPH and normal subjects. In particular, the CSF level assay of LRG suggests that LRG is a specific biomarker for INPH and has potential use in the diagnosis and indication for CSF shunting.

Stage-specific isoforms of Ascaris suum complex II: the fumarate reductase of the parasitic adult and the succinate dehydrogenase of free-living larvae share a common iron-sulfur subunit.

Complex II of adult Ascaris suum muscle exhibits high fumarate reductase (FRD) activity and plays a key role in anaerobic electron-transport during adaptation to their microaerobic habitat. In contrast, larval (L2) complex II shows a much lower FRD activity than the adult enzyme, and functions as succinate dehydrogenase (SDH) in aerobic respiration. We have reported the stage-specific isoforms of complex II in A. suum mitochondria, and showed that at least the flavoprotein subunit (Fp) and the small subunit of cytochrome b (cybS) of the larval complex II differ from those of adult. In the present study, complete cDNAs for the iron-sulfur subunit (Ip) of complex II, which with Fp forms the catalytic portion of complex II, have been cloned and sequenced from anaerobic adult A. suum, and the free-living nematode, Caenorhabditis elegans. The amino acid sequences of the Ip subunits of these two nematodes are similar, particularly around the three cysteine-rich regions that are thought to comprise the iron-sulfur clusters of the enzyme. The Ip from A. suum larvae was also characterized because Northern hybridization showed that the adult Ip is also expressed in L2. The Ip of larval complex II was recognized by the antibody against adult Ip, and was indistinguishable from the adult Ip by peptide mapping. The N-terminal 42 amino acid sequence of Ip in the larval complex II purified by DEAE-cellulofine column chromatography was identical to that of the mature form of the adult Ip. Furthermore, the amino acid composition of larval Ip determined by micro-analysis on a PVDF membrane is almost the same as that of adult Ip. These results, together with the fact, that homology probing by RT-PCR, using degenerated primers, failed to find a larval-specific Ip, suggest that the two different stage-specific forms of the A. suum complex II share a common Ip subunit, even though the adult enzyme functions as a FRD, while larval enzyme acts as an SDH.

Discovery of proteinaceous N-modification in lysine biosynthesis of Thermus thermophilus

Although the latter portion of lysine biosynthesis, the conversion of alpha-aminoadipate (AAA) to lysine, in Thermus thermophilus is similar to the latter portion of arginine biosynthesis, enzymes homologous to ArgA and ArgJ are absent from the lysine pathway. Because ArgA and ArgJ are known to modify the amino group of glutamate to avoid intramolecular cyclization of intermediates, their absence suggests that the pathway includes an alternative N-modification system. We reconstituted the conversion of AAA to lysine and found that the amino group of AAA is modified by attachment to the gamma-carboxyl group of the C-terminal Glu54 of a small protein, LysW; that the side chain of AAA is converted to the lysyl side chain while still attached to LysW; and that lysine is subsequently liberated from the LysW-lysine fusion. The fact that biosynthetic enzymes recognize the acidic globular domain of LysW indicates that LysW acts as a carrier protein or protein scaffold for the biosynthetic enzymes. This study thus reveals the previously unknown function of a small protein in primary metabolism.

Comprehensive proteomics analysis of autophagy-deficient mouse liver.

Autophagy is a bulk protein degradation system for the entire organelles and cytoplasmic proteins. Previously, we have shown the liver dysfunction by autophagy deficiency. To examine the pathological effect of autophagy deficiency, we examined protein composition and their levels in autophagy-deficient liver by the proteomic analysis. While impaired autophagy led to an increase in total protein mass, the protein composition was largely unchanged, consistent with non-selective proteins/organelles degradation of autophagy. However, a series of oxidative stress-inducible proteins, including glutathione S-transferase families, protein disulfide isomerase and glucose-regulated proteins were specifically increased in autophagy-deficient liver, probably due to enhanced gene expression, which is induced by accumulation of Nrf2 in the nuclei of mutant hepatocytes. Our results suggest that autophagy deficiency causes oxidative stress, and such stress might be the main cause of liver injury in autophagy-deficient liver.