Threonine-rich Region Research Articles

Ser/Thr-rich repetitive motifs as targets for phosphoglycan modifications in Leishmania mexicana secreted acid phosphatase.

The insect stage of the protozoan parasite Leishmania mexicana secretes a phosphomonoesterase in the form of a filamentous complex. The polypeptide subunits of this polymer are modified by phosphoglycans and/or oligomannosyl residues linked to phosphoserine. Based on peptide sequence data of a predominant 100 kDa protein of the filamentous complex, two tandemly arranged, single copy genes, lmsap1 and lmsap2, were cloned and sequenced. lmsap1 predicts a protein with features characteristic of acid phosphatases and a remarkable serine- and threonine-rich region of 32 amino acids close to the C-terminus. In the otherwise identical lmsap2 product, this region is extended to 383 amino acids and is composed of short Ser/Thr-rich repeats. Deletion analysis demonstrates that lmsap1 encodes the major 100 kDa protein of the complex while a minor 200 kDa component is derived from the lmsap2 gene. Null mutants of either gene retain the ability to secrete acid phosphatase filaments, while a deletion of both genes results in Leishmania defective in enzyme formation. The Ser/Thr-rich domains are the targets for phosphoglycan modifications as shown by the expression of secreted fusion proteins composed of these C-terminal regions and the N-terminal domain of a lysosomal acid phosphatase.

Biochemical and molecular characterization of the Pseudomonas lemoignei polyhydroxyalkanoate depolymerase system.

Pseudomonas lemoignei has five different polyhydroxyalkanoate (PHA) depolymerase genes (phaZ1 to phaZ5), which encode the extracellularly localized poly(3-hydroxybutyrate) (PHB) depolymerases C, B, and D, poly(3-hydroxyvalerate) (PHV) depolymerase, and PHB depolymerase A, respectively. Four of the five genes (phaZ1 to phaZ4) have been cloned, and one of them (phaZ1) was studied in detail earlier (D. Jendrossek, B. Müller, and H. G. Schlegel, Eur. J. Biochem. 218:701-710, 1993). The fifth PHA depolymerase gene (phaZ5) was identified by colony hybridization of recombinant Escherichia coli clones with a phaZ5-specific oligonucleotide. The nucleotide sequence of a 3,704-bp EcoRI fragment was determined and found to contain two large open reading frames (ORFs) which coded for a polypeptide with significant similarities to glycerol-3-phosphate dehydrogenases of various sources (313 amino acids; M(r), 32,193) and for the precursor of PHB depolymerase A (PhaZ5; 433 amino acids; M(r), 44,906). The PHV depolymerase gene (phaZ4) was subcloned, and the nucleotide sequence of a 3,109-bp BamHI fragment was determined. Two large ORFs (ORF3 and ORF4) that represent putative coding regions were identified. The deduced amino acid sequence of ORF3 (134 amino acids; M(r), 14,686) revealed significant similarities to the branched-chain amino acid aminotransferase (IlfE) of enterobacteria. ORF4 (1,712 bp) was identified as the precursor of a PHV depolymerase (567 amino acids; M(r), 59,947). Analysis of primary structures of the five PHA depolymerases of P. lemoignei and of the PHB depolymerases of Alcaligenes faecalis and Pseudomonas pickettii revealed homologies of 25 to 83% to each other and a domain structure: at their N termini, they have typical signal peptides of exoenzymes. The adjacent catalytic domains are characterized by several conserved amino acids that constitute putative catalytic triads which consist of the consensus sequence of serine-dependent hydrolases including the pentapeptide G-X-S-X-G, a conserved histidine and aspartate, and a conserved region resembling the oxyanion hole of lipases. C terminal of the catalytic domain an approximately 40-amino-acid-long threonine-rich region (22 to 27 threonine residues) is present in PhaZ1, PhaZ2, PhaZ3, and PhaZ5. Instead of the threonine-rich region PhaZ4 and the PHB depolymerases of A. faecalis and P. pickettii contain an approximately 90-amino-acid-long sequence resembling the fibronectin type III module of eucaryotic extracellular matrix proteins. The function of the fibronectin type III module in PHA depolymerases remains obscure. Two types of C-terminal sequences apparently represent substrate-binding sites; the PHB type is present in the PHB depolymerases of A. faecalis and P. pickettii and in PhaZ2, PhaZ3, and PhaZ5 and the PHV type is present in the PHV-hydrolyzing depolymerases (PhaZ4 and PhaZ1). phaZ1 was transferred to A. eutrophus H16 and JMP222. All transconjugants of both strains were able to grow with extracellular PHB as a carbon source and produced translucent halos on PHB-containing solid media. PhaZ1, PhaZ2, PhaZ4, and PhaZ5 were purified from P. lemoignei and from recombinant E. coli; the processing sites of the precursors in E. coli were the same as in P. lemoignei, and similar substrate specificities were determined for the wild-type and the recombinant proteins. All PHA depolymerases hydrolyzed PHB at high specific activities. PhaZ1 and PhaZ4 additionally cleaved PHV, and PhaZ4 hydrolyzed poly(4-hydroxybutyrate). None of the depolymerases was able to hydrolyze polyactide or PHA consisting of monomers with more than five carbon atoms. While the wild-type depolymerase proteins were glycosylated and found to contain glucose and N-acetylglucosamine, none of the recombinant proteins was glycosylated. PHB hydrolysis was dependent on divalent cations such as Ca2+ and was inhibited by the presence of EDTA.

Xylem-specific gene expression in loblolly pine

Two genes preferentially expressed in differentiating xylem of loblolly pine (Pinus taeda L.) were cloned from cDNA and genomic libraries and designated PtX3H6 and PtX14A9. Transcripts of PtX3H6 and PtX14A9 are very abundant in differentiating xylem, less abundant in needles, and very low or non-detectable in embryos and megagametophytes. PtX3H6 contains a putative signal peptide, a threonine-rich region, a proline-rich region, and a hydrophobic tail. Repeats of Pro-Pro-Pro-Val-X-X are similar to repeats found in proline-rich cell wall proteins. The amino acid compositions of PtX3H6 and PtX14A9 are similar to those of arabinogalactan proteins (AGPs). PtX14A9 contains an 8 amino acid sequence similar to amino terminal sequences of ryegrass, carrot and rose AGPs. Upstream sequences have been determined from genomic clones encoding PtX3H6 and PtX14A9. A 7 bp sequence found in the 5' flanking regions of both genes has previously been shown to be involved in the vascular-specific expression of GRP 1.8, a glycine-rich protein found in bean. The sequence is also present upstream of another glycine-rich protein from bean, GRP 1.0, and may be partially responsible for the xylem-specific expression of pTx3H6 and PtX14A9.

The Drosophila l(2)35Ba/nocA gene encodes a putative Zn finger protein involved in the development of the embryonic brain and the adult ocellar structures.

The Drosophila l(2)35Ba/nocA gene is involved in the development of the adult ocelli and the embryonic head. Mutations in this gene lead to at least two distinct phenotypes. Several larva lethal l(2)35Ba alleles cause both hypertrophy and mislocation of the embryonic supraesophageal ganglion (brain) to the dorsal surface of the embryo. A second class of mutant alleles (nocA) is homozygous viable, but the surviving adults either lack or have greatly reduced ocelli and associated bristles. The l(2)35Ba/nocA gene encodes an approximately 3.0-kb transcript doublet; all l(2)35Ba alleles which have been physically mapped delete or disrupt the transcribed region, whereas all of the viable nocA alleles are caused by gross chromosomal aberrations with breakpoints near the 3'-flanking region of the gene. Several nocA breakpoint alleles downregulate the level of l(2)35Ba/nocA transcripts in adults, and their defective ocellar phenotype also fails to be complemented by the lethal alleles, implying that l(2)35Ba and nocA are different phenotypic manifestations of mutations in the same gene. In the l(2)35Ba mutant embryos, cells from the procephalic lobe which normally migrate over and overlie the supraesophageal ganglion during head involution can become incorporated into the supraesophageal ganglion; many of these misplaced cells, which normally form the frontal sac, also adopt a neuronal fate. Sequence analysis of two full-length l(2)35Ba/nocA cDNAs with distinct polyadenylation sites shows that they encode the same deduced protein of 537 amino acids with a serine- and threonine-rich N-terminal region, two putative zinc finger motifs near the carboxyl terminus, and several alanine-rich domains. Consistent with the observed embryonic phenotype, l(2)35Ba/nocA shows a complex embryonic expression pattern which includes the procephalic lobe.

The Drosophila l(2)35Ba/nocA gene encodes a putative Zn finger protein involved in the development of the embryonic brain and the adult ocellar structures.

The Drosophila l(2)35Ba/nocA gene is involved in the development of the adult ocelli and the embryonic head. Mutations in this gene lead to at least two distinct phenotypes. Several larva lethal l(2)35Ba alleles cause both hypertrophy and mislocation of the embryonic supraesophageal ganglion (brain) to the dorsal surface of the embryo. A second class of mutant alleles (nocA) is homozygous viable, but the surviving adults either lack or have greatly reduced ocelli and associated bristles. The l(2)35Ba/nocA gene encodes an approximately 3.0-kb transcript doublet; all l(2)35Ba alleles which have been physically mapped delete or disrupt the transcribed region, whereas all of the viable nocA alleles are caused by gross chromosomal aberrations with breakpoints near the 3'-flanking region of the gene. Several nocA breakpoint alleles downregulate the level of l(2)35Ba/nocA transcripts in adults, and their defective ocellar phenotype also fails to be complemented by the lethal alleles, implying that l(2)35Ba and nocA are different phenotypic manifestations of mutations in the same gene. In the l(2)35Ba mutant embryos, cells from the procephalic lobe which normally migrate over and overlie the supraesophageal ganglion during head involution can become incorporated into the supraesophageal ganglion; many of these misplaced cells, which normally form the frontal sac, also adopt a neuronal fate. Sequence analysis of two full-length l(2)35Ba/nocA cDNAs with distinct polyadenylation sites shows that they encode the same deduced protein of 537 amino acids with a serine- and threonine-rich N-terminal region, two putative zinc finger motifs near the carboxyl terminus, and several alanine-rich domains. Consistent with the observed embryonic phenotype, l(2)35Ba/nocA shows a complex embryonic expression pattern which includes the procephalic lobe.

Structure of the sea urchin hatching enzyme gene

The sea urchin embryo develops from an encased to a free-living larva by secreting at an early stage the hatching enzyme, a metalloprotease which hydrolyses a protective envelope derived from the egg extracellular matrix. Genomic clones containing the entire hatching enzyme gene were isolated from a lambda phage sea urchin library and the complete sequence of the transcription unit was determined. The hatching enzyme gene spans 6.3 kb and comprises 9 exons. The exon/intron organization of the hatching enzyme gene is similar but not identical to those of the vertebrate collagenases and stromelysins. The position and/or phase of several introns are different even in the N-terminal moiety where similarity between echinoderm and vertebrate enzymes was first detected. The active-center domain is encoded by a 1-1 class exon whose sequence, length and borders are highly conserved and might be considered as coding for a protein module. Adjacent to the active-center exon, the hatching enzyme gene has an additional 1-1 exon which codes for a threonine-rich region. This provides further evidence that the matrix-degrading metalloproteinases evolved by shuffling exons of the 1-1 class. Phylogeny analysis indicates a close relationship between the sea urchin and vertebrate enzymes.

Sequence of a cDNA encoding the β 4 subunit of murine integrin

A cDNA coding for the β 4 subunit of murine integrin (mβ 4) has been cloned and sequenced using mRNA from a murine lung carcinoma as the template. The 5' sequence contains two AUG codons, the second of which initiates synthesis of the mature protein. The cDNA sequence has an open reading frame coding for 1748 amino acids (aa), including a signal peptide, cysteine-rich region, serine- and threonine-rich region, transmembrane domain, and a cytoplasmic domain of over 1000 aa. Overall, the deduced mβ4 aa sequence has 88% identity with the human β 4 subunit (hβ 4) sequence deduced from the sequence of placental mRNA. Reverse transcriptase-polymerase chain reaction using primers flanking splice sites for two variant forms of hβ 4 transcripts provided evidence for alternate splicing of RNA in the murine spleen and to a lesser extent in the skin, uterus, and thymus but was found at only one of the two alternative sites. Five potential glycosylation sites present in the extracellular domain of hβ 4 are conserved in mβ 4. One tyrosine in the terminal region of the cytoplasmic domain (position 1600) is conserved between mβ 4 and hβ 4 and has the consensus sequence for tyrosine phosphorylation. Finally, a genomic restriction map of mβ 4 shows that the gene is about 40 kb in length. No restriction-fragment length polymorphisms were detected between BALB/c liver and BALB/c lung carcinoma DNA.

Biochemical characterization of the extracellular domain of the 75-kilodalton tumor necrosis factor receptor.

An expression plasmid encoding the extracellular domain of the 75-kDa human tumor necrosis factor (TNF) type 2 receptor (TNF-R2) was constructed and used to generate a stable cell line secreting soluble TNF-R2 (sTNF-R2). Purified sTNF-R2 was resolved by SDS-PAGE into one band of approximate M(r) 43,000, consistent with a molecular weight of 36,000 +/- 4800 obtained by sedimentation equilibrium analysis. The apparent molecular weight observed by gel filtration chromatography was approximately 136,000. Glycosylation analysis revealed that Asn-149 is fully glycosylated, while Asn-171 is incompletely glycosylated (approximately 50%), and that a proline-, serine-, and threonine-rich region (residues 175-234) contains O-linked carbohydrate structures. Scatchard analysis of [125I]TNF-alpha and [125I]TNF-beta binding to sTNF-R2 gave dissociation constants (Kd) of 0.3 and 0.75 nM, respectively, comparable to those observed for intact cell-surface TNF-R2. The sTNF-R2 was found to block the cytotoxicity of both TNF-alpha and TNF-beta in a murine L-M cell assay. The sizes of the sTNF-R2.TNF-alpha and sTNF-R2.TNF-beta complexes determined by gel filtration chromatography were approximately 322 and 204 kDa, respectively. The stoichiometry of the sTNF-R2.TNF-alpha and sTNF-R2.TNF-beta complexes were examined by size-exclusion chromatography, sedimentation equilibrium, and cross-linking. The data from these studies suggest that at least two molecules of sTNF-R2 can bind to a single TNF-alpha or TNF-beta trimer.

Characterization of the Streptococcus mutans GS-5 fruA gene encoding exo-beta-D-fructosidase.

The complete nucleotide sequence (5,010 bp) of the fructanase gene (fruA) and flanking regions of the chromosome of Streptococcus mutans GS-5 was determined. The fruA gene appears to be the sole transcript arising from a proximal promoter. The presumed precursor of the secreted FruA protein consists of 1,423 amino acids, and it has an M(r) of 158,656 and a pI of 4.82. The N terminus of FruA has characteristics in common with signal peptides of gram-positive organisms. The C terminus consists of a serine- and threonine-rich region, followed by the peptide LPDTGD, 4 charged amino acids, 21 amino acids with a strongly hydrophobic character, and a charged pentapeptide tail, which are proposed to correspond to the wall-spanning region, the LPXTGX consensus sequence, and the membrane-spanning domains of surface-associated proteins of gram-positive cocci. The FruA protein has significant homology with the Bacillus subtilis levanase (SacC), the Bacteroides fragilis levanase (ScrL), yeast invertases, and a number of other beta-fructosidases but not with fructosyltransferase, glucosyltransferases, or glucan-binding proteins of oral streptococci. Genes with homology to fruA were detected in S. mutans serotype c, e, and f strains, Streptococcus rattus, Streptococcus salivarius, and Streptococcus sanguis. A deletion derivative of FruA lacking the C-terminal 437 amino acids was still functional and could hydrolyze beta-(2,6)- and beta-(2,1)-linked sugars, but with altered preference for substrates. The data begin to define functional domains of the FruA protein and potential regulatory sites for induction, repression, growth rate control, and posttranslational localization of this multifunctional enzyme.

EndAFS, a novel family E endoglucanase gene from Fibrobacter succinogenes AR1

The complete nucleotide sequence of endAFS, an endoglucanase gene isolated from the ruminal anaerobe Fibrobacter succinogenes AR1, was determined. endAFS encodes two overlapping open reading frames (ORF1 and ORF2), and it was proposed that a -1 ribosomal frameshift was required to allow contiguous synthesis of a 453-amino-acid endoglucanase. A proline- and threonine-rich region at the C terminus of ORF1 and rare codons for arginine and threonine were coincident with the proposed frameshift site. ENDAFS is proposed to be a member of subgroup 1 of family E endoglucanases, of which endoglucanases from Thermomonospora fusca and Persea americana (avocado) are also members. Endoglucanases from Clostridium thermocellum and Pseudomonas fluorescens form subgroup 2.

DNA sequence of a Fibrobacter succinogenes mixed-linkage beta-glucanase (1,3-1,4-beta-D-glucan 4-glucanohydrolase) gene

The DNA sequence of a mixed-linkage beta-glucanase (1,3-1,4-beta-D-glucan 4-glucanohydrolase [EC 3.2.1.73]) gene from Fibrobacter succinogenes cloned in Escherichia coli was determined. The general features of this gene are very similar to the consensus features for other gram-negative bacterial genes. The gene product was processed for export in E. coli. There is a high level of sequence homology between the structure of this glucanase and the structure of a mixed-linkage beta-glucanase from Bacillus subtilis. The nonhomologous region of the amino acid sequence includes a serine-rich region containing five repeats of the sequence Pro-Xxx-Ser-Ser-Ser-Ser-(Ala or Val) which may be functionally related to the serine-rich region observed in Pseudomonas fluorescens cellulase and the serine- and/or threonine-rich regions observed in Cellulomonas fimi endoglucanase and exoglucanase, in Clostridium thermocellum endoglucanases A and B, and in Trichoderma reesei cellobiohydrolase I, cellobiohydrolase II, and endoglucanase I.

Deletion of clustered O-linked carbohydrates does not impair function of low density lipoprotein receptor in transfected fibroblasts.

A single exon in the gene for the receptor for plasma low density lipoprotein (LDL) encodes a region of clustered serine and threonine residues that is immediately external to the membrane-spanning sequence. This region has been proposed as the site of clustered O-linked carbohydrate chains. In the current studies we have deleted the 144 base pairs (48 amino acids) that encode this serine- and threonine-rich region from the cDNA for the human LDL receptor. Upon transfection into receptor-deficient hamster fibroblasts, this mutated cDNA encoded a shortened receptor that no longer showed an anomalously high molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Labeling with [3H]glucosamine confirmed the lack of clustered O-linked sugars and further revealed that the shortened receptor and the normal receptor both contained isolated O-linked carbohydrate chains attached to the NH2-terminal portion of the protein. The ratio of clustered to isolated O-linked sugar chains in the normal receptor was estimated to be approximately 4-6 to 1. Despite the loss of clustered O-linked carbohydrate, the LDL receptor encoded by the deletion-bearing cDNA bound and internalized LDL normally. It also recycled normally and exhibited a normal half-life. We conclude that: 1) the serine- and threonine-rich region of the LDL receptor is the site for addition of clustered O-linked carbohydrates; 2) the receptor contains a small number of isolated chains of O-linked carbohydrates in addition to the clustered chains; and 3) the clustered O-linked carbohydrates are not essential for LDL receptor function in cultured hamster fibroblasts.