A monoclonal antibody, 3A10, recognizes a specific amino acid sequence present on a series of developmentally expressed brain proteins

Immunoblotting showed that a monoclonal antibody, 3A10, binds to a series of rat brain-specific antigens with molecular masses of 150-, 120-, 118-, 106-, 104-, 79-, and 77-kDa. The expression of 3A10 antigens is dependent on the developmental stage of the brain; only the 106-kDa antigen is detected during embryonic stages of rat brain development, while the expression of the remaining 6 antigens starts after birth and reaches a maximum during postnatal days 15-21. Detection of the 3A10 antigens in cultured neuronal and glial cells derived from cerebral cortices of rat brain at embryonic day 18 showed that the 77-, 79-, 106-, and 150-kDa antigens are specifically expressed in neuronal cells. The 77-kDa antigen was purified and identified as synapsin I by amino acid sequence analyses of the peptide fragments isolated after Achromobacter protease I treatment. During the isolation of 3A10-reactive proteins by immunological screening of cDNA libraries constructed from adult rat brain, we found that all of the 3A10-reactive clones contain nucleotide sequences encoding the unique amino acid sequence TRSP(S, R,G)P. Analyses of 3A10-binding to various synthetic peptides showed that the monoclonal antibody recognizes a specific conformational structure formed by either the TRSPXP sequence or similar amino acid sequences that are expressed on a series of developmentally expressed brain proteins.     

Type F scavenger receptor SREC-I interacts with advillin, a member of the gelsolin/villin family, and induces neurite-like outgrowth

The scavenger receptor expressed by endothelial cells (SREC) was isolated from a human endothelial cell line and consists of two isoforms named SREC-I and -II. Both isoforms have no significant homology to other types of scavenger receptors. They contain 10 repeats of epidermal growth factor-like cysteine-rich motifs in the extracellular domains and have unusually long C-terminal cytoplasmic domains with Ser/Pro-rich regions. The extracellular domain of SREC-I binds modified low density lipoprotein and mediates a homophilic SREC-I/SREC-I or heterophilic SREC-I/SREC-II trans-interaction. However, the significance of large Ser/Pro-rich cytoplasmic domains of SRECs is not clear. Here, we found that when SREC-I was overexpressed in murine fibroblastic L cells, neurite-like outgrowth was induced, indicating that the receptor can lead to changes in cell morphology. The SREC-I-mediated morphological change required the cytoplasmic domain of the protein, and we identified advillin, a member of the gelsolin/villin family of actin regulatory proteins, as a protein binding to this domain. Reduction of advillin expression in L cells by RNAi led to the absence of the described SREC-I-induced morphological changes, indicating that advillin is a prerequisite for the change. Finally, we demonstrated that SREC-I and advillin were co-expressed and interacted with each other in dorsal root ganglion neurons during embryonic development and that overexpression of both SREC-I and advillin in cultured Neuro-2a cells induced long process formation. These results suggest that the interaction of SREC-I and advillin are involved in the development of dorsal root ganglion neurons by inducing the described morphological changes.     

Purification and cDNA cloning of UDP-GlcNAc:GlcNAcbeta1-3Galbeta1-4Glc(NAc)-R [GlcNAc to Gal]beta1,6N-acetylglucosaminyltransferase from rat small intestine: a major carrier of dIGnT activity in rat small intestine

A rat intestinal beta1,6N-acetylglucosaminyltransferase (beta1-6GnT) responsible for the formation of the beta1,6-branched poly-N-acetyllactosamine structure has been purified to apparent homogeneity by successive column chromatographic procedures using an assay wherein pyridylaminated lacto- N-triose II (GlcNAcbeta1-3Galbeta1-4Glc-PA) was used as an acceptor substrate and the reaction product was GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-4Glc-PA. The purified enzyme catalyzed the conversion of the polylactosamine acceptor GlcNAcbeta1-3'LacNAc into GlcNAcbeta1-3'(GlcNAcbeta1-6') LacNAc (dIGnT activity), but it could not transfer GlcNAc to LacNAcbeta1-3'LacNAc (cIGnT activity). This enzyme could also convert mucin core 1 and core 3 analogs, Galbeta1-3GalNAcalpha1-O-paranitrophenyl (pNP) and GlcNAcbeta1-3GalNAcalpha1-O-pNP, into Galbeta1-3(GlcNAcbeta1-6) GalNAcalpha1-O-pNP (C2GnT activity) and GlcNAcbeta1-3(GlcNAcbeta1-6)GalNAcalpha1-O-pNP (C4GnT activity), respectively. Based on the partial amino acid sequences of the purified protein, the cDNA encoding this enzyme was cloned. The COS-1 cells transiently transfected with this cDNA had high dI/C2/C4GnT activities in a ratio of 0.34:1.00:0.90, compared with non- or mock-transfected cells. The primary structure shows a significant homology with human and viral mucin-type core 2 beta1-6GnTs (C2GnT-Ms), indicating that this enzyme is the rat ortholog of human and viral C2GnT-Ms. This is the first identification and purification of this enzyme as a major carrier of dIGnT activity in the small intestine. This rat ortholog should mostly be responsible for making distal I-branch structures on poly-N-acetyllactosamine sequences in this tissue, as well as making mucin core 2 and core 4 structures, given that it also has high C2/C4GnT activities.     

Kinesin transports RNA: isolation and characterization of an RNA-transporting granule

RNA transport is an important and fundamental event for local protein synthesis, especially in neurons. RNA is transported as large granules, but little is known about them. Here, we isolated a large RNase-sensitive granule (size: 1000S approximately) as a binding partner of conventional kinesin (KIF5). We identified a total of 42 proteins with mRNAs for CaMKIIalpha and Arc in the granule. Seventeen of the proteins (hnRNP-U, Pur alpha and beta, PSF, DDX1, DDX3, SYNCRIP, TLS, NonO, HSPC117, ALY, CGI-99, staufen, three FMRPs, and EF-1alpha) were extensively investigated, including their classification, binding combinations, and necessity for the "transport" of RNA. These proteins and the mRNAs were colocalized to the kinesin-associated granules in dendrites. The granules moved bidirectionally, and the distally directed movement was enhanced by the overexpression of KIF5 and reduced by its functional blockage. Thus, kinesin transports RNA via this granule in dendrites coordinately with opposite motors, such as dynein.     

Purification,properties, and partial amino acid sequences of alanine racemase from the muscle of the black tiger prawn Penaeus monodon

Alanine racemase [EC 5.1.1.1], which catalyzes the interconversion between D- and L-alanine, was purified to homogeneity from the muscle of black tiger prawn Penaeus monodon. The isolated enzyme had a molecular mass of 44 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 90 kDa on gel filtration, indicating a dimeric nature of the enzyme. The enzyme was highly specific to D- and L-alanine and did not catalyze the racemization of other amino acids. K(m) values toward both D- and L-alanine were almost equal and considerably high compared with those of bacterial enzymes. The purified enzyme retained its activity in the absence of pyridoxal 5'-phosphate as a cofactor but carbonyl reagents inhibited the activity, suggesting the tightly binding of the cofactor to the enzyme protein. Several partial amino acid sequences of peptide fragments of the purified enzyme showed positive homologies from 52 to 76% with bacterial counterparts and a catalytic tyrosine residue of the bacterial enzyme was also retained in the prawn one, indicating alanine racemase gene is well conserved from bacteria to invertebrates.     

Production of homo- and hetero-dimeric isozymes from two aldehyde oxidase genes of Arabidopsis thaliana.

Polyclonal antibodies were raised against synthetic peptides or recombinant polypeptides encoded by Arabidopsis atAO-1 and atAO-2 cDNAs, which have sequences similar to maize and animal aldehyde oxidase (AO) cDNAs. Anti-atAO-1 antibodies recognized AOalpha and AObeta among the three isoforms, AOalpha, AObeta, and AOgamma, detected in Arabidopsis seedlings after native PAGE, while anti-atAO-2 antibodies reacted with AObeta and AOgamma. The polypeptide specifically recognized by each antibody was collected as the Protein-A/IgG/antigen complex. The 150- and 145-kDa polypeptides were purified by SDS-PAGE and digested with Achromobacter Protease I. From the amino acid sequences and molecular masses of the derivative peptides, it was revealed that the 150- and 145-kDa polypeptides were the products of atAO-1 and atAO-2, respectively. Molecular masses of the native forms of AOalpha, AObeta, and AOgamma were estimated as approximately 290-300 kDa. These results suggest that AOalpha and AOgamma are homodimers consisting of atAO-1 and atAO-2 products, respectively, and that AObeta is a heterodimer of the atAO-1 and atAO-2 products.