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Y P Loh

Publications and source records attributed to Y P Loh.

At least 19 recordsLinked to original sources
PubMed1999

Identification and characterization of Saccharomyces cerevisiae yapsin 3, a new member of the yapsin family of aspartic proteases encoded by the YPS3 gene.

A new aspartic protease from Saccharomyces cerevisiae, with a high degree of similarity with yapsin 1 and yapsin 2 and a specificity for basic residue cleavage sites of prohormones, has been cloned. This enzyme was named yapsin 3. Expression of a C-terminally truncated non-membrane anchored yapsin 3 in yeast yielded a heterogeneous protein between 135-200 kDa which, upon treatment with endoglycosidase H, migrated as a 60 kDa form. Amino-acid analysis of the N-terminus of expressed yapsin 3 revealed two different N-terminal residues, serine-48 and phenylalanine-54, which followed a dibasic and a monobasic residue respectively. Cleavage of several prohormones by non-anchored yapsin 3 revealed a specificity distinct from that of yapsin 1.

Amino Acid Sequence↗
PubMed1999

Identification of a novel prohormone sorting signal-binding site on carboxypeptidase E, a regulated secretory pathway-sorting receptor.

Sorting of the prohormone POMC to the regulated secretory pathway necessitates the binding of a sorting signal to a sorting receptor, identified as membrane carboxypeptidase E (CPE). The sorting signal, located at the N terminus of POMC consists of two acidic (Asp10, Glu14) and two hydrophobic (Leu11, Leu18) residues exposed on the surface of an amphipathic loop. In this study, molecular modeling of CPE predicted that the acidic residues in the POMC-sorting signal bind specifically to two basic residues, Arg255 and Lys260, present in a loop unique to CPE, compared with other carboxypeptidases. To test the model, these two residues on CPE were mutated to Ser or Ala, followed by baculovirus expression of the mutant CPEs in Sf9 cells. Sf9 cell membranes containing CPE mutants with either Arg255 or Lys260, or both residues substituted, showed no binding of [125I]N-POMC1-26 (which contains the POMC-sorting signal motif), proinsulin, or proenkephalin. In contrast, substitution of an Arg147 to Ala147 at a substrate-binding site, Arg259 to Ala259 and Ser202 to Pro202, in CPE did not affect the level of [125I]N-POMC1-26 binding when compared with-wild type CPE. Furthermore, mutation of the POMC-sorting signal motif (Asp10, Leu11, Glu14, Leu18) eliminated binding to wild-type CPE. These results indicate that the sorting signal of POMC, proinsulin, and proenkephalin specifically interacts with Arg255 and Lys260 at a novel binding site, independent of the active site on CPE.

Animals↗
PubMed1998

Carboxypeptidase E is a sorting receptor for prohormones: binding and kinetic studies.

The binding of pro-opiomelanocortin,(POMC), pro-insulin, pro-enkephalin and chromogranin A (CGA) to the regulated secretory pathway sorting receptor, carboxypeptidase E (CPE), in bovine pituitary secretory granule (SG) membranes was investigated. N-POMC1-26, which contains the POMC sorting signal, bound to CPE in the SG membranes with low affinity and the binding was ion independent. Pro-insulin bound CPE with similar kinetics. Pro-enkephalin, but not CGA bound to CPE with similar IC50 as pro-insulin and N-POMC1-26. Crosslinking studies showed that pro-insulin and pro-enkephalin bound specifically to SG membrane CPE, similar to N-POMC1-26 reported previously. CPE was extracted from the SG membranes with NaHCO3 or KSCN, but not Triton X-100/1 M NaCl. The results show that CPE is tightly associated with SG membranes and binds several prohormones, but not CGA, with similar kinetics, providing further evidence that membrane CPE has the characteristics to function as a common sorting receptor for targeting prohormones to the regulated secretory pathway.

Animals↗
PubMed1998

Cleavage efficiency of the novel aspartic protease yapsin 1 (Yap3p) enhanced for substrates with arginine residues flanking the P1 site: correlation with electronegative active-site pockets predicted by molecular modeling.

Yapsin 1, a novel aspartic protease with unique specificity for basic residues, was shown to cleave CCK13-33 at Lys23. Molecular modeling of yapsin 1 identified the active-site cleft to have negative residues close to or within the S6, S3, S2, S1, S1', S2', and S3' pockets and is more electronegative than rhizopuspepsin or endothiapepsin. In particular, the S2' subsite has three negative charges in and close to this pocket that can provide strong electrostatic interactions with a basic residue. The model, therefore, predicts that substrates with a basic residue in the P1 position would be favored with additional basic residues binding to the other electronegative pockets. A deletion of six residues close to the S1 pocket in yapsin 1, relative to rhizopuspepsin and other aspartic proteases of known 3D structure, is likely to affect its specificity. The model was tested using CCK13-33 analogues. We report that yapsin 1 preferentially cleaves a CCK13-33 substrate with a basic residue in the P1 position since the substrates with Ala in P1 were not cleaved. Furthermore, the cleavage efficiency of yapsin 1 was enhanced for CCK13-33 analogues with arginine residues flanking the P1 position. An alanine residue, substituting for the arginine residue in the P6 position in CCK13-33, resulted in a 50% reduction in the cleavage efficiency. Substitution with arginine residues downstream of the cleavage site at the P2', P3', or P6' position increased the cleavage efficiency by 21-, 3- and 7-fold, respectively. Substitution of Lys23 in CCK13-33 with arginine resulted not only in cleavage after the substituted arginine residue, but also forced a cleavage after Met25, suggesting that an arginine residue in the S2' pocket is so favorable that it can affect the primary specificity of yapsin 1. These results are consistent with the predictions from the molecular model of yapsin 1.

Alanine↗
PubMed1998

Activation and processing of non-anchored yapsin 1 (Yap3p).

A C-terminally truncated form of yapsin 1 (yeast aspartic protease 3), the first member of the novel sub-class of aspartic proteases with specificity for basic residues (designated the Yapsins), was overexpressed and purified to apparent homogeneity, yielding approximately 1 microg of yapsin 1/g of wet yeast. N-terminal amino acid analysis of the purified protein confirmed that the propeptide was absent and that the mature enzyme began at Ala68. The mature enzyme was shown to be composed of approximately equimolar amounts of two subunits, designated alpha and beta, that were associated to each other by a disulfide bond. C-terminally truncated proyapsin 1 was also expressed in the baculovirus/Sf9 insect cell expression system and secreted as a zymogen that could be activated upon incubation at an acidic pH with an optimum at approximately 4.0. When expressed without its pro-region, it was localized intracellularly and lacked activity, indicating that the pro-region was required for the correct folding of the enzyme. The activation of proyapsin 1 in vitro exhibited linear kinetics and generated an intermediate form of yapsin 1 or pseudo-yapsin 1.

Amino Acid Sequence↗
PubMed1998

Vasopressin gene expression in rat choroid plexus.

Vasopressin (VP) levels in cerebrospinal fluid (CSF) change in response to physiological stimuli and under various pathological conditions. The sources of CSF VP have yet to be clarified, however. In the present study, we provide evidence indicating that VP is synthesized in the choroid plexus, the primary site of CSF formation. All experiments were performed on adult male Sprague-Dawley rats. The presence of VP mRNA in choroid plexus epithelium was demonstrated by in situ hybridization histochemistry using the 35S-labeled riboprobe that was complementary to cDNA fragment of rat VP encoding the C-terminus part of proVP. In situ hybridization findings were confirmed by reverse transcriptase-polymerase chain reaction analysis. Immunohistochemistry for VP-associated neurophysin (VP-NP), a polypeptide component of proVP, revealed subapical accumulation of VP-NP-immunopositive product in choroidal epithelial cells. Immunoprecipitation and immunoblotting of choroidal protein extracts with anti-VP-NP antibody demonstrated the presence of a approximately 10-kD polypeptide that was also detected in hypothalamus. We hypothesize that the choroid plexus-derived VP exerts autocrine and/or paracrine effects on tissues near the CSF system.

Animals↗
PubMed1998

Depletion of carboxypeptidase E, a regulated secretory pathway sorting receptor, causes misrouting and constitutive secretion of proinsulin and proenkephalin, but not chromogranin A.

Previous studies have shown that the prohormone POMC is sorted to the regulated secretory pathway (RSP), at the trans-Golgi network, by binding of a conformation-dependent sorting signal to a sorting receptor, identified as membrane-bound carboxypeptidase E (CPE) (Cool et al., 1997, Cell, 88:73-83). In this study, the role of CPE as a sorting receptor for other RSP proteins that contain sorting signals (proinsulin, proenkephalin, and chromogranin A) was investigated in neuroendocrine cells (Neuro-2a) stably expressing CPE antisense RNA. Whereas these cells were depleted of CPE by greater than 85%, electron microscopy showed that they contain dense core secretory granules identical to wild-type Neuro-2a cells, indicating that CPE is not essential for granulogenesis. Secretion and immunocytochemical studies showed that, in wild-type Neuro-2a cells, endogenous proenkephalin and transfected proinsulin/insulin were localized to punctate secretory granules and were released via the RSP. However, in CPE-depleted cells, these two prohormones were released constitutively and had a Golgi-like distribution but were not localized to punctate secretory granules. In contrast, chromogranin A was present in punctate secretory granules and released via the RSP, in wild-type and CPE-depleted Neuro-2a cells. Thus, the sorting of proinsulin and proenkephalin to the RSP, like POMC, necessitates binding to CPE, and hence, CPE acts as a common sorting receptor for targeting these prohormones to the RSP. In contrast, the sorting signal of chromogranin A does not use CPE as a sorting receptor, suggesting the existence of other sorting receptors for the RSP.

Carboxypeptidase H↗
PubMed1997

High-performance liquid chromatographic-colorimetric assay for glycine carboxypeptidase activity.

A rapid and sensitive assay method for the determination of glycine carboxypeptidase activity has been reported. This method is based on the monitoring of the absorption at 460 nm of 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe, enzymatically formed from the substrate 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe-Gly, after separation by high-performance liquid chromatography (HPLC) using a TSK gel ODS-80TM reversed-phase column by isocratic elution. This method is sensitive enough to measure 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe at concentrations as low as 1 pmol and yield highly reproducible results and requires less than 7.5 min per sample for separation and quantitation. The pH optimum for glycine carboxypeptidase activity was 4.8 to 5.4. The Km and Vmax values were respectively 21.1 micromol and 3.73 pmol/microg/h with the use of enzyme extract obtained from bovine pituitary. Glycine carboxypeptidase activity was strongly inhibited by Ag+, Cu2+ and p-chloromercuriphenylsulfonic acid. Among the organs examined in a mouse, the highest specific activity of the enzyme was found in testis. The sensitivity and selectivity of this method will aid in efforts to examine the physiological role of this peptidase.

Animals↗
PubMed1997

Intracellular misrouting and abnormal secretion of adrenocorticotropin and growth hormone in cpefat mice associated with a carboxypeptidase E mutation.

Cpefat mice carry a mutation in the carboxypeptidase E/H gene which encodes an exopeptidase that removes C-terminal basic residues from endoproteolytically cleaved hormone intermediates. These mice have endocrine disorders including obesity, infertility, and hyperproinsulinemia-diabetes syndrome, but the etiology remains an enigma. Because studies have identified membrane carboxypeptidase E as a sorting receptor for targeting prohormones to the regulated secretory pathway for processing and secretion, the intracellular routing and secretion of pro-opiomelanocortin/adrenocorticotropin and growth hormone from anterior pituitary cells were investigated in Cpefat mice. In Cpefat mice, pro-opiomelanocortin was accumulated 24-fold above normal animals in the pituitary and it was poorly processed to adrenocorticotropin. Furthermore, pro-opiomelanocortin was secreted constitutively at high levels, showing no response to stimulation by corticotropin-releasing hormone. Similarly, growth hormone release was constitutive and did not respond to high K+ stimulation. Both pro-opiomelanocortin and growth hormone levels were elevated in the circulation of Cpefat mice versus normal mice. These data provide evidence that the lack of carboxypeptidase E, the sorting receptor, results in the intracellular misrouting and secretion of pro-opiomelanocortin and growth hormone via the constitutive pathway in the pituitary of Cpefat mice.

Adrenocorticotropic Hormone↗
PubMed1997

The structure of synenkephalin (pro-enkephalin 1-73) is dictated by three disulfide bridges.

Mass spectrometry of fragments produced by limited proteolytic digestion of pro-enkephalin was used to locate the disulfide bridges in synenkephalin (pro-enkephalin 1-73), a domain which contains sorting information for targeting the pro-neuropeptide to the granules of the regulated secretory pathway in neuroendocrine cells. Mass spectrometric analysis was optimized by using chemicals that gave low interference with the ionization and desorption processes, and computer software which simplified the identification of all possible disulfide-linked peptide fragments. Three disulfide bridges between Cys2-Cys24, Cys6-Cys28, and Cys9-Cys41 were identified. Protein conformational prediction of synenkephalin1-42 shows beta-turns which facilitate the formation of these disulfide bonds.

Amino Acid Sequence↗
PubMed1997

Carboxypeptidase E is a regulated secretory pathway sorting receptor: genetic obliteration leads to endocrine disorders in Cpe(fat) mice.

A proposed mechanism for sorting secretory proteins into granules for release via the regulated secretory pathway in endocrine-neuroendocrine cells involves binding the proteins to a sorting receptor at the trans-Golgi network, followed by budding and granule formation. We have identified such a sorting receptor as membrane-associated carboxypeptidase E (CPE) in pituitary Golgi-enriched and secretory granule membranes. CPE specifically bound regulated secretory pathway proteins, including prohormones, but not constitutively secreted proteins. We show that in the Cpe(fat) mutant mouse lacking CPE, the pituitary prohormone, pro-opiomelanocortin, was missorted to the constitutive pathway and secreted in an unregulated manner. Thus, obliteration of CPE, the sorting receptor, leads to multiple endocrine disorders in these genetically defective mice, including hyperproinsulinemia and infertility.

Amino Acid Sequence↗
PubMed1997

Enhanced brain opioid receptor activity precedes blood-brain barrier disruption.

We studied the effects of transient postischemic increased opioid receptors (OPR) binding (mu, delta, kappa) on blood-brain barrier (BBB), brain water content and brain mitochondrial oxidative enzymes system. Cats were exposed to temporary middle cerebral artery occlusion (MCAO). The significant increased OPR bindings observed 10 min after the release of MCAO (ischemic rCBF = 7 +/- 1 to 11 +/- 2 ml/100 g/min) preceded the early and late BBB disruptions, brain edema and postischemic impaired mitochondrial oxidative enzymes functions. Further, the study suggests indirectly that the latter process was irreversible and hence associated with subsequent ischemic cerebral infarction. In addition, the results revealed a possible viable therapeutic window in the early postischemic recirculation period, before the onset of impaired mitochondrial oxidative function.

Animals↗
PubMed1997

The presence of arginine vasopressin and its mRNA in rat choroid plexus epithelium.

Arginine vasopressin (AVP) plays an important role in the regulation of secretory function and hemodynamics of choroid plexus, the primary site of cerebrospinal fluid (CSF) production. In the present study, localization of AVP and its transcripts in choroid plexus of adult male Sprague-Dawley rats was studied by immunohistochemistry and in situ hybridization histochemistry, respectively. For immunohistochemical analysis, AVP-specific polyclonal rabbit antibody was employed. Plasmid, pGrVP, containing a 232-bp fragment of rat AVP cDNA encoding the C-terminus of proAVP, was used as a probe to detect AVP mRNA. AVP-immunoreactive product was predominantly localized close to the apical (CSF-facing) membrane of choroidal epithelium while AVP transcripts were distributed throughout the cytoplasm of the cells. Our findings indicate that AVP is synthesized in choroid plexus epithelium, which suggests autocrine and/or paracrine actions of this peptide in choroidal tissue.

Animals↗
PubMed1996

Yeast aspartic protease 3 (Yap3) prefers substrates with basic residues in the P2, P1 and P2' positions.

The yeast aspartic protease Yap3 is localised to the secretory pathway and correctly cleaves pro-alpha-mating factor at its dibasic sites. We determined the specificity of Yap3 for mono-, di-, and multi-basic cleavage sites in the context of 15 residue synthetic proalbumin peptides. Yap3 cleaved after dibasic ArgArg and LysArg sites but not after monobasic Arg sites even when there was an additional arginine at -6 and/or -4. Yap3 did not cleave a tetra-arginine site and tri-basic sites (RRR and RRK) were poor substrates. Cleavage always occurred C-terminal to the last arginine in the di- or tri-basic sequence. The optimal cleavage site sequence was RR DR and this substrate was cleaved 8-9-fold faster than the normal RR DA sequence. In contrast to Kex2, Yap3 did not remove the propeptide from normal proalbumin or a range of natural or recombinant proalbumin variants. However at pH 4.0 Yap3 slowly cleaved proalbumin and albumin between domains 2 and 3.

Amino Acid Sequence↗
PubMed1996

Specificity and kinetic studies on the cleavage of various prohormone mono- and paired-basic residue sites by yeast aspartic protease 3.

The specificity and relative efficiency of cleavage of mono- and paired-basic residue processing sites by YAP3p was determined in vitro for a number of prohormone substrates: human ACTH1 39, bovine proinsulin, porcine cholecystokinin 33, cholecystokinin (CCK) 13-33, dynorphin A(1-11), dynorphin B(1-13), and amidorphin. YAP3p generated ACTH1-15 from ACTH1-39. It cleaved proinsulin at the paired-basic residue sites of the B-C junction as well as the C-A junction. Leu-enkephalin-Arg and Leu-enkephalin-Arg-Arg were generated from dynorphin A and dynorphin B, respectively. YAP3p generated Met-enkephalin-Lys-Lys from amidorphin showing that cleavage by this enzyme can occur at a lone pair of Lys residues. CCK33 was cleaved at Lys23 and Arg9, each containing an upstream Arg residue at the P6 and P5 position, respectively. Km values were between 10(-4) and 10(-5) M for the various substrates, with the highest affinity exhibited for the tetrabasic site of ACTH1-39 (1.8 x 10(-5) M). The tetrabasic residue site of ACTH1-39 was cleaved with the highest relative efficiency (kcat/Km = 3.1 x 10(6) m-1 s-1), while that of the monobasic site of CCK13-33 and the paired-basic site of proinsulin B-C junction, were cleaved less efficiently at 4.2 x 10(4) m-1 s-1 and 1.6 x 10(4) m-1 s-1, respectively.

Adrenocorticotropic Hormone↗
PubMed1996

Processing of pro-opiomelanocortin in GH3 cells: inhibition by prohormone convertase 2 (PC2) antisense mRNA.

The processing of pro-opiomelanocortin (POMC) was examined in GH3 cells, a rat sommatomammotrope cell line, by transiently-transfecting the cells with mouse POMC cDNA. The peptide products were extracted, chromatographed on HPLC and identified by specific radioimmunoassay. POMC was processed to generate ACTH-related peptides, beta-endorphin and Lys-gamma 3- MSH, with complete disappearance of the POMC precursor. The ACTH-related molecules were identified as ACTH1-14, ACTH1-15, ACTH1-17, as well as ACTH1-39. GH3 cells which were not transfected with POMC cDNA did not contain endogenous POMC-related peptides. RT-PCR demonstrated that GH3 cells contain prohormone convertase 2 (PC2) mRNA but no PC1 mRNA. To determine if PC2 was the enzyme responsible for POMC processing in this cell line, GH3 cells were stably-transfected with PC2 antisense cDNA. A cell line was obtained which showed an absence of PC2 protein compared to control untransfected GH3 cells, indicating successful hybridization of PC2 antisense mRNA to the endogenous PC2 mRNA. When this cell line was then transiently-transfected with POMC cDNA, POMC was not processed. The results from these experiments suggest that PC2 alone can correctly process POMC to biologically active smaller peptides in vivo. Additionally, the GH3 cell line with and without incorporation of PC2 antisense cDNA can be used as a model system to study the role of PC2 in the post-translational processing of other prohormones and proproteins in vivo.

Animals↗