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PubMed1994

A twelve year natural history of hepatitis C virus infection in hemodialyzed patients.

A prospective non-A, non-B follow-up program, implemented in a hepatitis B surface antigen-free dialysis unit, enabled us to report on the natural history of hepatitis C virus (HCV) infection in hemodialyzed patients between 1980 and 1992. For this program, every patient was prospectively monitored every two weeks for alanine amino transferase (ALT) activity, and every month for gammaglutamyl transpeptidase (GGT) activity and systematic collection of frozen sera. Sequences of stored sera from 217 patients were repeatedly tested for anti-HCV antibodies using second generation assays. Eighty-six of the 217 patients (39.6%), including 61 of the 67 patients with non-A, non-B hepatitis (91%), had HCV infection repeatedly evidenced by positive ELISA in all, and confirmed by RIBA in 84 of 86 (97.5%). In addition, 19 out of 23 patients (82.6%) were positive for HCV RNA by the polymerase chain reaction (PCR). Of the 86 anti-HCV positive patients, 41 had previously acquired HCV infection, and 45 seroconverted during chronic dialysis. Of these, all but one patient developed hepatitis with raised ALT activity which lasted for at least six months in all. Only 29 of 45 patients (64.5%) had a history of blood transfusion. Seventy-eight of the 86 patients (91%) who were followed up for one to 11.5 years (median 5) retained anti-HCV for several years. Nineteen liver biopsies performed in 16 patients showed chronic active hepatitis in 8 (50%) and hepatocellular carcinoma without cirrhosis in one patient.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine Transaminase↗

PubMed2000

Multiple nuclear pseudogenes of mitochondrial cytochrome b in Ctenomys (Caviomorpha, rodentia) with either great similarity to or high divergence from the true mitochondrial sequence.

A fragment of the mitochondrial cytochrome b gene was studied in 13 species of the South American fossorial rodent Ctenomys using PCR with 'universal' primers and DNA sequencing after cloning. Five different groups of sequences were found, one of which corresponds to the functional mitochondrial gene (mt). The other four groups (A, B, C and D) were believed to be nuclear pseudogenes. Sequences A-C were highly divergent from the mt sequences and included substitutions, deletions and insertions such that they could not possibly have coded a functional protein. They all shared a common insertion between positions 15055 and 15056 suggestive of a common origin, although the A, B and C sequences otherwise differed greatly from each other. The D sequences also could not have been functional on the basis of nucleotide sequence, but the differences with the mt sequences were far more subtle and in a more limited study the D sequences could easily have been classified as a true mtDNA sequence. It is suggested that there were two transfers of the cytochrome b gene from the mitochondrion to the nucleus; the first leading to sequences A-C and the second to the D sequence. Subsequent to transfer, a sequence of duplications within the nucleus appears to have generated the full range of pseudogenes that are observed. This study adds to other recent observations suggesting the frequent transfer of mtDNA sequences to the nucleus and reinforces the necessity of great care in interpreting PCR-generated sequences, particularly those produced with universal primers. There are now data from several species of mammals and birds relating to PCR-generated nuclear copies of cytochrome b, which we review.

Animals↗

PubMed1988

Isolation and primary structure of tumor-derived peptides related to human pancreastatin and chromogranin A.

Using an antiserum raised against a synthetic C-terminal peptide of porcine pancreastatin, we detected pancreastatin-like immunoreactivity (PLI) in human pancreatic islets, adrenal medulla, and endocrine tumors. From a carcinoid liver metastasis, human PLI was extracted and purified by HPLC. Two C-terminally amidated peptides were isolated and characterized by sequence analysis. The first peptide, hCgA-210-301, consists of 92 amino acid residues with glycinamide as C terminus. It is identical to the cDNA-derived sequence of human chromogranin A, positions 210-301, which is preceded by two basic residues indicating a putative processing site. The C-terminal part, positions 250-301, shows 70% sequence identity to porcine pancreastatin and represents the human pancreastatin-like sequence. The second peptide, hCgA-273-301, represents a C-terminally amidated fragment of the human pancreastatin sequence, generated by an Asp-Pro cleavage at the N terminus. Peptide hCgA-273-301 was synthesized to confirm the structure of the natural peptide. Two other peptides derived from human chromogranin A were isolated and partially characterized. They are generated by proteolytic cleavage after dibasic amino acids Lys-Arg (positions 338-339) and after Trp-376 of the human chromogranin A sequence, respectively. These results indicate that chromogranin A may represent the precursor for pancreastatin-related and possibly other yet-unidentified peptides of unknown physiological function.

Amino Acid Sequence↗

PubMed2000

Embedded solitons in a three-wave system

We report a rich spectrum of isolated solitons residing inside (embedded into) the continuous radiation spectrum in a simple model of three-wave spatial interaction in a second-harmonic-generating planar optical waveguide equipped with a quasi-one-dimensional Bragg grating. An infinite sequence of fundamental embedded solitons is found, each one differing by the number of internal oscillations. Branches of these zero-walkoff spatial solitons give rise, through bifurcations, to several secondary branches of walking solitons. The structure of the bifurcating branches suggests a multistable configuration of spatial optical solitons, which may find straightforward applications for all-optical switching.

Journal Article↗

PubMed1978

Cyanide-insensitive NADH oxidation by subcellular fractions isolated from human polymorphonuclear blood cells.

The biochemical triad, NADH oxidation, oxygen (O2) uptake and hydrogen peroxide (H2O2) formation, by subcellular fractions of human blood polymorphonuclears (PMNs) was investigated. It was found that this biochemical triad (1) was under the control of the granule-rich fraction (GRF) only; (2) was not inhibited by cyanide; (3) occurred stoichiometrically for its three components, and (4) accounted quantitatively for the respiratory burst of the stimulated PMN. It was also shown that the above biochemical triad (1) involved an enzymatic step; (2) was enhanced by acidic pH (0.5) and Mg++; (3) was inhibited by Cu++ or low concentration of Mn++; (4) was dependent on H2O2, perhydroxyl radical (HO2) and hydroxyl radical (HO) since either catalase or superoxide dismutase or scavengers of HO2 or HO were inhibitor, and (5) involved multistep reactions. Evidence is provided that the sequence of the reactions is first a generation of H2O2, (spontaneously from NADH in our incubation medium), secondly the production of HO from H2O2, thirdly the oxidation of NADH with further production of HO2,O2 uptake and H2O2 formation, probably through a chain reaction. The identification of the enzyme(s) involved in these multistep reactions needs further studies.

Cyanides↗

PubMed2004

Generation and analysis of expressed sequence tags from Trypanosoma cruzi trypomastigote and amastigote cDNA libraries.

We have generated 2771 expressed sequence tags (ESTs) from two cDNA libraries of Trypanosoma cruzi CL-Brener. The libraries were constructed from trypomastigote and amastigotes, using a spliced leader primer to synthesize the cDNA second strand, thus selecting for full-length cDNAs. Since the libraries were not normalized nor pre-screened, we compared the representation of transcripts between the two using a statistical test and identify a subset of transcripts that show apparent differential representation. A non-redundant set of 1619 reconstructed transcripts was generated by sequence clustering. This dataset was used to perform similarity searches against protein and nucleotide databases. Based on these searches, 339 sequences could be assigned a putative identity. One thousand one-hundred and sixteen sequences in the non-redundant clustered dataset (68.8%) are new expression tags, not represented in the T. cruzi epimastigote ESTs that are in the public databases. Additional information is provided online at http://genoma.unsam.edu.ar/projects/tram. To the best of our knowledge these are the first ESTs reported for the life cycle stages of T. cruzi that occur in the vertebrate host.

Animals↗

PubMed1991

Escherichia coli molecular genetic map (1500 kbp): update II.

The DNA sequence data for Escherichia coli deposited in the EMBL library (release 27), together with miscellaneous data obtained from several laboratories, have been localized on an updated and corrected version of the restriction map of the chromosome generated by Kohara et al. (1987) and modified by others. This second update adds a further 500 kbp, increasing the amount of the E. coli chromosome sequenced to about one third of the total: 1510 kbp of sequenced DNA is included in the present data base. The accuracy of the map is assessed, and allows us to propose a precise genetic map position for every sequenced gene. The location of rare-cutting sites such as AvrII, NotI and SfiI have also been included in the update in order to combine the data obtained from different sources into one single file. The distribution of palindromic sequences (to which most restriction sites belong) has been studied in coding sequences. There appears to be a significant counter-selection against several such sequences in E. coli coding sequences (but not in other organisms such as Saccharomyces cerevisiae), suggesting the existence of constraints on DNA structure in E. coli, perhaps indicative of a functional role for horizontal gene transfer, preserving coding sequences, in this type of bacteria.

Base Sequence↗

PubMed1994

Subcellular locations of MOD5 proteins: mapping of sequences sufficient for targeting to mitochondria and demonstration that mitochondrial and nuclear isoforms commingle in the cytosol.

MOD5, a gene responsible for the modification of A37 to isopentenyl A37 of both cytosolic and mitochondrial tRNAs, encodes two isozymes. Initiation of translation at the first AUG of the MOD5 open reading frame generates delta 2-isopentenyl pyrophosphate:tRNA isopentanyl transferase I (IPPT-I), which is located predominantly, but not exclusively, in the mitochondria. Initiation of translation at a second AUG generates IPPT-II, which modifies cytoplasmic tRNA. IPPT-II is unable to target to mitochondria. The N-terminal sequence present in IPPT-I and absent in IPPT-II is therefore necessary for mitochondrial targeting. In these studies, we fused MOD5 sequences encoding N-terminal regions to genes encoding passenger proteins, pseudomature COXIV and dihydrofolate reductase, and studied the ability of these chimeric proteins to be imported into mitochondria both in vivo and in vitro. We found that the sequences necessary for mitochondrial import, amino acids 1 to 11, are not sufficient for efficient mitochondrial targeting and that at least some of the amino acids shared by IPPT-I and IPPT-II comprise part of the mitochondrial targeting information. We used indirect immunofluorescence and cell fractionation to locate the MOD5 isozymes in yeast. IPPT-I was found in two subcellular compartments: mitochondria and the cytosol. We also found that IPPT-II had two subcellular locations: nuclei and the cytosol. The nuclear location of this protein is surprising because the A37-->isopentenyl A37 modification had been predicted to occur in the cytoplasm. MOD5 is one of the first genes reported to encode isozymes found in three subcellular compartments.

Alkyl and Aryl Transferases↗

PubMed1994

Development of 124 sequence-tagged sites and cytogenetic localization of 217 cosmids for human chromosome 10.

A total of 124 new chromosome 10-specific sequence-tagged sites (STSs) were derived from two sources: (1) DNA sequences obtained from anonymous clones in new libraries enriched for human chromosome 10 inserts, and (2) published sequences of genes and other loci already known to map to chromosome 10. Libraries were constructed from a somatic cell hybrid carrying human chromosomes 10 and Y. A cosmid library was made from total DNA of the hybrid and probed with labeled total human DNA to identify clones with human DNA inserts. Two hundred seventeen cosmids were mapped to regions of human chromosome 10 by fluorescence in situ hybridization. Twenty-five cosmids represent probes that have been placed on the genetic map previously. One hundred ninety-two cosmids represent new probes that have not been mapped previously. Cosmids carrying inserts with CA repeats were identified by hybridization with a labeled poly(dC-dA)-poly(dG-dT) probe and subcloned to yield microsatellite STS markers. Two small insert plasmid libraries were made, the first by subcloning inserts from a chromosome 10-enriched lambda phage library (LL10NS01) and the second by cloning Alu element-mediated PCR products amplified from hybrid DNA. STSs were generated from the DNA sequences of clone inserts. Chromosome 10-specific STSs were distinguished from Y chromosome STSs by one or both of the following criteria: (1) successful PCR amplification from a template consisting of DNA from another chromosome 10-containing cell line, NA10926B, or (2) FISH localization to chromosome 10 of the source cosmid or of YACs isolated by PCR screening with the STS. These libraries were the source of 90 new chromosome 10-specific STSs, 42 of which contain CA repeats.

Base Sequence↗

PubMed1994

Determinants of carboxyl-terminal domain translocation during prion protein biogenesis.

The prion protein (PrP) displays some unusual features in its biogenesis. In cell-free systems it can be synthesized as either an integral transmembrane protein spanning the membrane twice, with both amino and carboxyl domains in the lumen of the endoplasmic reticulum, or as a fully translocated polypeptide. A charged, extracytoplasmic region, termed the Stop Transfer Effector (STE) sequence, has been shown to direct the nascent translocating chain to stop at the adjoining hydrophobic domain to generate the first membrane-spanning region (TM1). However, the determinants of the second translocation event in the biogenesis of the transmembrane form have not been identified previously. Moreover, the relationship of transmembrane and fully translocated forms of PrP has not been well understood. Here, we report progress in resolving both of these issues. Using protein chimeras in cell-free translation systems and Xenopus oocytes, we identify the sequence which directs nascent PrP to span the membrane a second time, with its carboxyl-terminal domain in the endoplasmic reticulum lumen. Surprisingly, PrP carboxyl-terminal domain translocation does not appear to be directed by an internal signal or signal-anchor sequence located downstream of TM1, as would have been expected from studies of other multispanning membrane proteins. Rather, carboxyl-terminal domain translocation appears to be another consequence of the action of STE-TM1, that is, the same sequence responsible for generating the first membrane-spanning region. Studies of an STE-TM1-containing protein chimera in Xenopus oocytes demonstrate that most of these chains upon completion of their translation, initially span the membrane twice, with a topology similar to that of transmembrane PrP, but are carbonate-extractable. These chains have the transmembrane orientation only transiently and chase into a fully translocated form. These results support a model in which a metastable "transmembrane" intermediate, residing within the aqueous environment of the translocation channel, can be converted into either the integrated transmembrane or the fully translocated form of PrP, perhaps directed by trans-acting factor (s). Such a model may explain why stable the transmembrane isoform of PrP has not been observed in normal cells and how nascent PrP might be directed to alternate pathways of folding.

Amino Acid Sequence↗

PubMed2005

Domain-selective ligand-binding modes and atomic level pharmacophore refinement in angiotensin I converting enzyme (ACE) inhibitors.

Somatic ACE (EC 3.4.15.1), a Zn(II) metalloproteinase, is composed of functionally active N and C domains resulting from tandem gene duplication. Despite the high degree of sequence similarity between the two domains, they differ in substrate and inhibitor specificity and in their activation by chloride ions. Because of the critical role of ACE in cardiovascular and renal diseases, both domains are attractive targets for drug design. Putative structural models have been generated for the interactions of ACE inhibitors (lisinopril, captoril, enalaprilat, keto-ACE, ramiprilat, quinaprilat, peridoprilat, fosinoprilat, and RXP 407) with both the ACE_C and the ACE_N domains. Inhibitor-domain selectivity was interpreted in terms of residue alterations observed in the four subsites of the binding grooves of the ACE_C/ACE_N domains (S1: V516/N494, V518/T496, S2: F391/Y369, E403/R381, S1': D377/Q355, E162/D140, V379/S357, V380/T358, and S2': D463/E431, T282/S260). The interactions governing the ligand-receptor recognition process in the ACE_C domain are: a salt bridge between D377, E162, and the NH(2) group (P1' position), a hydrogen bond of the inhibitor with Q281, the presence of bulky hydrophobic groups in the P1 and P2' sites, and a stacking interaction of F391 with a benzyl group in the P2 position. In ACE_N these interactions are: hydrogen bonds of the inhibitor with E431, Y369, and R381, and a salt bridge between the carboxy group in the P2 position of the inhibitor and R500. The calculated complexes were evaluated for their consistency with structure-activity relationships and site-directed mutagenesis data. A comparison between the calculated interaction free energies and the experimentally observed biological activities was also made. Pharmacophore refinement was achieved at an atomic level, and might provide an improved basis for structure-based rational design of second-generation, domain-selective inhibitors.

Amino Acid Sequence↗

PubMed1994

Plasma desorption mass spectrometry of two synthetic sarafotoxins: side reactions and characterization of the intermediates.

Sarafotoxins (SRTXs) form a family of toxic and potent vasoconstrictor peptides of 21 amino acids and two disulfide bonds. They are present in the venom of the burrowing asp Atractaspis engaddensis. We have made two derivatives of the amino acid sequence of SRTX-b, one of the most potent isotoxins, in the solid phase. First, we replaced Ser2 by Thr, to investigate whether, as previously postulated, this change is responsible for the weak activities of SRTXs c and d. Secondly, we replaced Ser2, Asp18 and Val19 respectively by Thr, Gly and Ile, with a view to generating SRTX-e whose amino acid sequence was deduced from cDNA. Solid-phase peptide synthesis (SPPS) was performed according to the tert-butyloxycarbonyl strategy and the disulfides were paired sequentially using a selective chemistry. The disulfide 1-15 was formed by oxidation of cysteines1,15 with ferricyanide, whereas disulfide 3-11 was made by iodine oxidation of Acm-blocked cysteines3,11. By plasma desorption mass spectrometry (PDMS), we monitored all possible side reactions that occurred during the synthesis. We thus observed a benzyl shift in mass spectra when aspartic and glutamic acid side chains were protected by a benzyl group during the SPPS. This could be circumvented by using instead, a cyclohexyl protecting group. We also noted the oxidation of the methionine and the tryptophan side chain (formation of methionine sulfoxide and oxindole ring of tryptophan) to a small extent during the cleavage peptide/solid phase oxidation of the methionine side chain during the formation of the disulfide 1-15 by ferricyanide.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed2005

Extracting stacking interaction parameters for RNA from the data set of native structures.

A crucial step in the determination of the three-dimensional native structures of RNA is the prediction of their secondary structures, which are stable independent of the tertiary fold. Accurate prediction of the secondary structure requires context-dependent estimates of the interaction parameters. We have exploited the growing database of natively folded RNA structures in the Protein Data Bank (PDB) to obtain stacking interaction parameters using a knowledge-based approach. Remarkably, the calculated values of the resulting statistical potentials (SPs) are in excellent agreement with the parameters determined using measurements in small oligonucleotides. We validate the SPs by predicting 74% of the base-pairs in a dataset of structures using the ViennaRNA package. Interestingly, this number is similar to that obtained using the measured thermodynamic parameters. We also tested the efficacy of the SP in predicting secondary structure by using gapless threading, which we advocate as an alternative method for rapidly predicting RNA structures. For RNA molecules with less than 700 nucleotides, about 70% of the native base-pairs are correctly predicted. As a further validation of the SPs we calculated Z-scores, which measure the relative stability of the native state with respect to a manifold of higher free energy states. The computed Z-scores agree with estimates made using calorimetric measurements for a few RNA molecules. Structural analysis was used to rationalize the success and failures of SP and experimentally determined parameters. First, from the near perfect linear relationship between the number of native base-pairs and sequence length, we show that nearly 46% of nucleotides are not in stacks. Second, by analyzing the suboptimal structures that are generated in gapless threading we show that the SPs and experimentally determined parameters are most successful in predicting stacks that end in hairpins. These results show that further improvement in secondary structure prediction requires reliable estimates of interaction parameters for loops, bulges, and stacks that do not end in hairpins.

Base Pairing↗

PubMed1993

Natural evolution of coronavirus defective-interfering RNA involves RNA recombination.

Defective-interfering (DI) RNAs of RNA viruses, in general, are generated and continue to evolve in size during serial undiluted passages of viruses. This evolution was thought to occur by independent generation of DI RNAs during virus passages and subsequent selection of new DI RNAs under new cellular conditions. Here we demonstrate that recombination between the old DI RNA and the helper viral RNA can be one of the mechanisms for natural DI RNA evolution. A mouse hepatitis virus (MHV) DI RNA, DIssE RNA, was transcribed in vitro and transfected into a mouse cell line infected with a different MHV strain (A59), which is distinguishable from the original natural helper MHV (JHM). During subsequent serial undiluted passages of the harvested virus, several novel DI RNA species were generated, while the original DIssE RNA disappeared by passage 11. cDNA cloning and sequence analysis of one of these novel DI RNAs, designated DI-2, revealed that it is composed of four discontinuous regions of the genomic sequence and is different from the structure of the original DIssE RNA. Sequence comparison among DI-2, DIssE, and helper MHV-A59 RNAs showed that DI-2 sequence is similar to DIssE in the first and second regions, but similar to the helper A59 virus in the third and last regions. Thus, this DI RNA was generated by RNA recombination between the original DIssE RNA and the helper viral RNA. These results indicate that recombination between DI RNA and helper virus RNA can be involved in the natural evolution of DI RNAs.

Animals↗

PubMed1990

Isolation, characterization, and mapping of a human acid beta-galactosidase cDNA.

A lambda gt11 human testicular cDNA library was screened with degenerate oligonucleotide probe mixtures based on amino acid sequence data generated from cyanogen bromide fragments and tryptic fragments of purified human beta-galactosidase. Six positive clones were identified after screening 2 x 10(6) plaques. The sequences of these six clones were determined and found to be derived from two different cDNAs. The sequence of the longest of these cDNAs is nearly identical to that recently determined by Oshima et al. (1988). It codes for a 76-kD protein and all 11 peptides that were generated from the purified enzyme. The second clone is shorter by 393 bp in the central portion of the coding region. Analysis by Northern blotting revealed the presence of a single mRNA species of 2.45 kb in lymphoblasts and testicular tissue. It is deduced from the amino acid sequence data that proteolytic processing of the precursor form of beta-galactosidase must occur by cleavage in the carboxy-terminal portion of the polypeptide perhaps around amino acid 530 at a uniquely hydrophilic sequence. Using a probe generated from the 3' region of the cDNA, we have mapped the locus coding for human beta-galactosidase to chromosome 3p21-3pter.

Amino Acid Sequence↗

PubMed2004

Post-transcription cleavage generates the 3' end of F17R transcripts in vaccinia virus.

Most vaccinia virus intermediate and late mRNAs possess 3' ends that are extremely heterogeneous in sequence. However, late mRNAs encoding the cowpox A-type inclusion protein (ATI), the second largest subunit of the RNA polymerase, and the late telomeric transcripts possess homogeneous 3' ends. In the case of the ATI mRNA, it has been shown that the homogeneous 3' end is generated by a post-transcriptional endoribonucleolytic cleavage event. We have determined that the F17R gene also produces homogeneous transcripts generated by a post-transcriptional cleavage event. Mapping of in vivo mRNA shows that the major 3' end of the F17R transcript maps 1262 nt downstream of the F17R translational start site. In vitro transcripts spanning the in vivo 3' end are cleaved in an in vitro reaction using extracts from virus infected cells, and the site of cleavage is the same both in vivo and in vitro. Cleavage is not observed using extract from cells infected in the presence of hydroxyurea; therefore, the cleavage factor is either virus-coded or virus-induced during the post-replicative phase of virus replication. The cis-acting sequence responsible for cleavage is orientation specific and the factor responsible for cleavage activity has biochemical properties similar to the factor required for cleavage of ATI transcripts. Partially purified cleavage factor generates cleavage products of expected size when either the ATI or F17R substrates are used in vitro, strongly suggesting that cleavage of both transcripts is mediated by the same factor.

Animals↗

PubMed1994

Differential expression and regulation of multiple dynamins.

Dynamin is a GTP-, microtubule-, and phospholipid-binding protein that is expressed primarily in brain. In Drosophila, the shibire gene encodes a homologue of dynamin; mutations in this gene result in a defect in endocytosis, suggesting a function for dynamin in endocytic membrane traffic. In the present study we show that there are at least two distinct dynamin genes in mammals whose products are referred to as dynamins I and II. The two dynamins are similar to each other (79% identity) and are both equally homologous to the Drosophila shibire gene product (66% identity). The highest degree of identity between dynamins is observed in their N-terminal halves, whereas their C termini exhibit little homology. Transcripts of both dynamin genes are subject to at least two alternative splicing events, the first of which is identically found in both dynamins, whereas the second site of alternative splicing is different between the two types of dynamins. The first alternatively spliced sequence of the dynamins consists of an interior region that is present in two distinct but homologous forms in both dynamins, suggesting alternative use of exons in both genes at identical positions. The second site of alternative splicing results in the generation of different C termini in dynamin I and in the inclusion or exclusion of an interior four-amino acid sequence in dynamin II. The two dynamins exhibit remarkable differences in their tissue distribution and regulation. Dynamin I is almost exclusively expressed in the central nervous system. Conversely, dynamin II is expressed ubiquitously in all tissues tested. Previous studies revealed that the GTPase activity of dynamin I is regulated by phosphorylation by protein kinase C in nerve terminals. Expression of dynamins I and II by transfection in COS cells demonstrates that only dynamin I but not dynamin II is a substrate for protein kinase C. Our data suggest a specialization in the endocytic functions and the regulation of dynamins between neural and non-neural tissues in mammals.

Amino Acid Sequence↗

PubMed1992

Cloning and primary structure of neurocan, a developmentally regulated, aggregating chondroitin sulfate proteoglycan of brain.

We have obtained the complete coding sequence of neurocan, a chondroitin sulfate proteoglycan of rat brain which is developmentally regulated with respect to its molecular size, concentration, carbohydrate composition, sulfation, and immunocytochemical localization. Two degenerate oligonucleotides, based on amino acid sequence data from the proteoglycan isolated from adult brain by immunoaffinity chromatography with the 1D1 monoclonal antibody, were used as sense and antisense primers in the polymerase chain reaction with a brain cDNA library as template to generate an unambiguous cDNA probe. A second probe for the N-terminal portion of the early postnatal form of the proteoglycan was obtained by reverse transcription/polymerase chain reaction. The composite sequence of overlapping cDNA clones is 5.2-kilobases (kb) long, including 1.3 kb of 3'-untranslated sequence and 76 base pairs of 5'-untranslated sequence. An open reading frame of 1257 amino acids encodes a protein with a molecular mass of 136 kDa containing 10 peptide sequences present in the adult and/or early postnatal brain proteoglycans. The deduced amino acid sequence revealed a 22-amino acid signal peptide followed by an immunoglobulin domain, tandem repeats characteristic of the hyaluronic acid-binding region of aggregating proteoglycans, and an RGDS sequence. The C-terminal portion (amino acids 951-1215) has approximately 60% identity to regions in the C termini of the fibroblast and cartilage proteoglycans, versican and aggrecan, including two epidermal growth factor-like domains, a lectin-like domain, and a complement regulatory protein-like sequence. The central 595-amino acid portion of neurocan has no homology with other reported protein sequences. The proteoglycan contains six potential N-glycosylation sites and 25 potential threonine O-glycosylation sites. In the adult form of the proteoglycan (which represents the C-terminal half of neurocan) a single 32-kDa chondroitin 4-sulfate chain is linked at serin-944, whereas three additional potential chondroitin sulfate attachment sites (only two of which are utilized) are present in the larger proteoglycan species. A probe corresponding to a region of neurocan having no homology with versican or aggrecan hybridized with a single band at approximately 7.5 kb on Northern blots of mRNA from both 4-day and adult rat brain (but not with muscle, kidney, liver, or lung mRNA), indicating that the 1D1 proteoglycan of adult brain, containing a 68-kDa core protein, is generated by a developmentally regulated in vivo proteolytic processing of the 136-kDa species which is predominant in early postnatal brain.(ABSTRACT TRUNCATED AT 400 WORDS)

Aggrecans↗