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PubMed1992

Expression in human lung tumor cells of the proprotein processing enzyme PC1/PC3. Cloning and primary sequence of a 5 kb cDNA.

Northern blot analysis of human lung tumors indicated that the gene, which encodes the subtilisin-like proprotein processing enzyme PC1/PC3, was highly expressed in almost all carcinoid tumors tested. In small cell lung carcinomas (SCLCs), expression varied. In non-SCLCs and normal lung, no expression was found. Analysis of SCLC cell lines revealed that expression was restricted preferentially to cell lines of the classical type. In lung tumor cells expressing the PC1/PC3 gene, transcripts of 3 kb and 5 kb were detected, the 5 kb mRNA always being the most abundant species. We isolated a cDNA corresponding to the 5 kb human PC1/PC3 transcript, determined the nucleotide sequence of it and deduced the amino acid sequence of the corresponding protein. Furthermore, we conclude that the two PC1/PC3 transcripts have 3' non-coding regions of different size and encode the same protein.

Amino Acid Sequence↗
PubMed1992

A modified Kex2 enzyme retained in the endoplasmic reticulum prevents disulfide-linked dimerisation of recombinant human insulin-like growth factor-1 secreted from yeast.

The majority of the recombinant human insulin-like growth factor-1 (IGF1) molecules, secreted from yeast using the prepro sequence of the prepro-alpha-factor, are not active monomers but inactive, disulfide-linked dimers. The prepro sequence of the prepro-alpha-factor, usually referred to as the alpha-factor leader (alpha FL), consists of a pre or signal sequence and a proregion. After signal sequence removal during translocation into the endoplasmic reticulum (ER) the proregion is still attached to IGF1 when it folds to acquire a tertiary structure. Mature IGF1 is released only in a late Golgi compartment by the membrane-bound endoprotease Kex2p. We find that co-expression of a novel ER-retained Kex2p variant, soluble Kex2pHDEL, can prevent intermolecular disulfide bond formation between two IGF1 molecules, implying that the presence of the proregion during the folding of IGF1 in the ER could be a reason for disulfide-linked dimerisation. This result indicates that the proregion of the alpha FL may have a role in the folding of some heterologous proteins in yeast, and that the ER-retained Kex2p mutant could be used as a convenient tool to study the cellular function of the proregions present naturally in various eucaryotic precursor proteins.

Blotting, Western↗
PubMed1993

Processing of Kex2 pro-region at two interchangeable cleavage sites.

In Saccharomyces cerevisiae, the Kex2 endoprotease (Kex2p) is required for the proteolytic maturation of alpha-pheromone and also for the removal of its own pro-region. Kex2p is specific for pairs of basic amino acid residues. Two putative processing sites are present in the pro-region of Kex2p. We have expressed processing site mutants of Kex2p and assayed the production of active Kex2p. Mutations affecting either putative cleavage site do not alter the activity. However, mutations affecting both sites led to a reduction in both Kex2 activity and the amount of protein. These results suggest that removal of Kex2p pro-peptide is required for the production of a stable enzyme and can occur at either processing site.

Enzyme Stability↗
PubMed1993

Localization of Kex2-like processing endoproteases, furin and PC4, within mouse testis by in situ hybridization.

By in situ hybridization analysis, we show here the localization of furin and PC4, which are both members of a growing family of endoproteases structurally related to the yeast precursor processing protease Kex2, within mouse testis. Furin transcript was detected in both germ and somatic cells, while PC4 transcript was found only in round spermatids. Proenkephalin transcript was also localized in round spermatids. These observations suggest that, within testis, PC4 is involved in processing of peptide precursors such as proenkephalin and may play a role in regulation of sperm maturation, while furin may serve as a more general processing endoprotease.

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

Limulus kexin: a new type of Kex2-like endoprotease specifically expressed in hemocytes of the horseshoe crab.

A Kex2-like protease was identified in hemocytes of the horseshoe crab (Tachypleus tridentatus), named limulus kexin, and a full-length cDNA was obtained from a hemocyte cDNA library. The deduced amino acid sequence contains 752 residues, composed of five domains with a signal sequence, a propeptide, a catalytic domain, a Ser/Thr-rich domain, and a transmembrane domain. The domain organization is very similar to that of the yeast Kex2 except that limulus kexin does not have a cytoplasmic tail. The catalytic domain exhibits striking sequence identities with those of furins, especially Drosophila furin1 (79%). Northern blotting showed specific expression of limulus kexin in hemocytes, suggesting the involvement in proteolytic processing of the granule components of hemocytes.

Amino Acid Sequence↗
PubMed1996

Donor-side photoinhibition in photosystem II from Chlamydomonas reinhardtii upon mutation of tyrosine-Z in the D1 polypeptide to phenylalanine.

When tyrosine-Z of the D1-polypeptide of the photosystem II from Chlamydomonas reinhardtii was changed to phenylalanine, the rapid donor to P680+ was lost, and P680+ accumulated on illumination. The rapid donation from tyrosine-Z was replaced by a slow electron transfer from an endogenous donor. Spectrophotometric measurements showed that carotenoids and chlorophylls were bleached by the P680+ either directly or indirectly upon illumination. The carotenoid bleaching was inhibited in the presence of SOD or catalase, but the reaction did not require molecular oxygen as an electron acceptor. These observations led us to conclude that active oxygen radicals, possibly hydroxyl radicals, take part in the destruction of carotenoids in the Y161F mutant. Possible mechanisms for the destruction are discussed.

Algal Proteins↗
PubMed1996

Determination of the disulfide array of the first inducible antifungal peptide from insects: drosomycin from Drosophila melanogaster.

Drosomycin is a 44-residue antifungal peptide with four intramolecular disulfide bridges which have been isolated from immune-challenged Drosophila. To produce adequate amounts of this peptide for 3D-structure analysis, studies on the mode of action and activity spectrum, we expressed a synthetic cDNA in Saccharomyces cerevisiae. For this purpose, we used the mating factor alpha gene and concomitantly overexpressed the KEX2 gene to increase the yield of fully processed drosomycin. Using a combination of Edman degradation and mass spectrometry, we show that drosomycin shares the same array of intramolecular disulfide bridges than plant defensins, in addition to their sequence similarities.

Amino Acid Sequence↗
PubMed1982

Processing of normal and non-glycosylated forms of toad pro-opiocortin by rat intermediate (pituitary) lobe pro-opiocortin converting enzyme activity.

The influence of glycosylation of a prohormone, pro-opiocortin, on its processing by intermediate (pituitary) lobe converting enzyme activity in vitro was studied. [3H]-arginine-labeled glycosylated and non-glycosylated pro-opiocortins were isolated from untreated, and tunicamycin treated toad neurointermediate lobes, respectively, after pulse-labeling in [3H]-arginine containing incubation media. These labeled precursors were then incubated at 37 degrees C in the presence of pro-opiocortin converting enzyme activity derived from rat intermediate lobe (pituitary) secretory granule lysates. The rates of conversion of the glycosylated and nonglycosylated pro-opiocortins to smaller peptide products, in vitro, were similar. Analysis of the peptide products by immunoprecipitation with ACTH and beta-endorphin antisera, and subsequent electrophoresis on acid-urea gels, indicate a comparable processing in vitro of the two forms of pro-opiocortin substrate. The only difference was that the normally glycosylated peptide products derived from glycosylated pro-opiocortin (i.e., 13K ACTH, 21K ACTH, and the 16K glycopeptide) differed in their gel electrophoretic mobilities from their counterparts derived from nonglycosylated prohormone, in a manner consistent with the absence of carbohydrate on the latter's peptides. These data show that glycosylation of the prohormone does not influence its processing in vitro by the converting enzyme activity.

Animals↗
PubMed1990

Arginine and lysine product inhibition of bovine adrenomedullary carboxypeptidase H, a prohormone processing enzyme.

Carboxypeptidase H (CPH) is one of the later enzymes in the cascade of proteolytic steps required for the posttranslational processing of peptide hormone precursors, including processing of proenkephalin. In this study, CPH activity in the soluble and membrane fractions of enkephalin-containing bovine chromaffin granules was competitively inhibited by its products arginine and lysine. Ki values for arginine and lysine were 4.6 +/- 1.3 and 7.6 +/- 1.9 mM, respectively, indicating that arginine was a more effective inhibitor than lysine. Other amino acids (at 10 mM) had no effect. The in vivo intragranular concentrations of lysine and arginine are similar to the measured Ki values, indicating that product inhibition of CPH by basic amino acids may occur in vivo.

Adrenal Medulla↗
PubMed1993

Characterization of pro-opiomelanocortin processing in heterologous neuronal cells that express PC2 mRNA.

We have investigated processing of monkey pro-opiomelanocortin (POMC) following transfection into heterologous neuronal Neuro 2A (N2A) cells. In several separately transfected stable cell lines (termed N2A/POMC2-like; n = 4), POMC was processed to beta E only, by direct cleavage from the precursor. Thus, these cell lines did not produce beta E in the orderly manner observed in the pituitary, that is, via the intermediate peptide beta LPH. Analysis of one representative N2A/POMC2 cell line revealed that the extent of processing to beta E appeared to be negatively correlated with precursor expression level, suggesting that the processing enzyme(s) in these cells was present in limiting amounts. Northern analysis of PC1 and PC2, two recently cloned processing enzymes, showed that N2A/POMC2 cells expressed low levels of PC2 mRNA, but no detectable PC1 mRNA. These data suggest that (1) the order of processing observed in the pituitary is not exclusively determined by tertiary folding of the precursor, but rather by the complement of processing enzymes in a particular cell, and (2) if PC2 is responsible for POMC processing in N2A/POMC2 cells, this enzyme, expressed in limiting amounts, appeared to show selectivity for the beta E amino terminal processing site.

Animals↗
PubMed1995

Expression of antisense PC1 in stably transfected RIN5F cells significantly reduces CCK 8 biosynthesis.

The subtilisin-like endoprotease PC1 (PC3) has been implicated in the processing of a number of prohormones. To evaluate whether PC1 may be important for the processing of pro CCK to CCK 8, stable cell lines expressing a portion of the PC1 cDNA in the antisense orientation were established from RIN5F cells. These cells express CCK mRNA, produce and display regulated secretion of CCK 8. One of the clones, R1E8, expresses antisense PC1 mRNA as determined by reverse transcriptase-PCR (RT-PCR) and contains a significantly reduced level of PC1 protein. As compared to both RIN5F and RIN5F control cells (transfected with the expression plasmid containing no antisense message), R1E8 contains only about 30% cell content of CCK 8. These results suggest that PC1 may be important for the processing of CCK 8 from pro CCK.

Animals↗
PubMed1996

In vivo processing of the precursor of the major exoglucanase by KEX2 endoprotease in the Saccharomyces cerevisiae secretory pathway.

We have established the main post-translational modification of the major exoglucanase of Saccharomyces cerevisiae as the enzyme progresses through the secretory pathway. The protein portion of the enzyme accumulated by sec18 cells was about 2 kDa larger than that of the secreted enzyme. This precursor (form A) was stable when maintained in the endoplasmic reticulum but was processed to the mature form (form B) before the block imposed by the sec7 mutation. Sec7 cells, when incubated at 37 degrees C, accumulated form B first, but upon prolonged incubation, form A was preferentially accumulated. When the supply of newly synthesized exoglucanase was prevented by the addition of cycloheximide, the accumulated A was transformed into B in the presence of altered Sec7p that still prevented secretion. Conversion of A into B was prevented in the double mutant sec7 kex2-1, indicating that Kex2p is central to the in vivo processing. Consistent with this, a KEX2 deletion mutant secreted form A exclusively. Conversion of A into B was also prevented in sec7 cells by the presence of dinitrophenol, a poison that depletes ATP levels, indicating that processing is dependent upon intracellular transport which involves ER --> Golgi and/or, at least, one intra-Golgi step(s). It follows that this transport step(s) is independent of functional Sec7p.

Adenosine Triphosphatases↗
PubMed1993

Secretion of heterologous proteins by Aspergillus niger: production of active human interleukin-6 in a protease-deficient mutant by KEX2-like processing of a glucoamylase-hIL6 fusion protein.

To develop improved methods for heterologous protein production in Aspergillus niger, we studied the secretion of human interleukin-6 (hIL6). Since in vitro experiments with culture medium revealed that hIL6 was rapidly degraded, several protease-deficient strains of A. niger were isolated and tested for reduced degradation of hIL6 compared with the wild-type strain. The mutant strain giving the least degradative effect on hIL6 (designated AB1.13) was transformed with several hIL6-expression plasmids. Initially, hIL6 was expressed using various signal sequences fused to the sequence of mature hIL6. The resulting transformants did not produce detectable amounts of hIL6, despite high transcription levels in one transformant. We hypothesized that hIL6 was not efficiently processed during passage along the secretion pathway. Therefore, hIL6 was expressed as a fusion protein with glucoamylase, a protein which is efficiently secreted by A. niger and expression of which can easily be measured enzymatically. To obtain mature hIL6, a sequence encoding the KEX2 cleavage-site (Lys-Arg) was inserted between glucoamylase and hIL6 sequences. Mature active hIL6 was found to be secreted in the extracellular medium. Using this combined approach of transforming a protease-deficient strain with a fusion construct containing the KEX2 site, up to 15 mg l-1 active hIL6 was obtained in shake-flask culture. A fusion construct without the KEX2 site resulted in substantially higher production of the fusion protein, but hIL6 was not active in the fused form. These results indicate that A. niger contains a protease with similar specificity as the KEX2 protease from yeast.

Amino Acid Sequence↗
PubMed1995

Production of bioactive enkephalin from the nonendocrine cell lines COS-7, NIH3T3, Ltk-, and C2C12.

Enkephalin is synthesized from proenkephalin in neuroendocrine cells. For the attempt to induce nonneuroendocrine origin cells to produce enkephalin, we used a mammalian expression vector for fusion peptides, pMEproCT beta, in which a fused peptide is designed to be cleaved by a yeast Kex2-like endoprotease furin. Met-Enkephalin was expressed in four nonneuroendocrine cell lines: COS-7, C2C12, Ltk-, and NIH3T3. The four cell lines produced a marked amount of Met-enkephalin, which appeared as a single peak on reverse-phase HPLC. Because transplantation of adrenal medullary cells to the subarachnoid space has been used to alleviate terminal cancer pain, and enkephalin appears to play a central role in relieving pain, this enkephalin expression vector may be useful for direct enkephalin expression in pericancerous tissues.

3T3 Cells↗