Generation of a biologically active, secreted form of human thyroid peroxidase by site-directed mutagenesis

Using site-directed mutagenesis, we introduced two stop codons immediately upstream of the putative transmembrane domain in human thyroid peroxidase (hTPO) cDNA, truncating the carboxyl terminus of hTPO (933 amino acids) by 85 residues. Mutated hTPO cDNA, inserted into a eukaryotic expression vector, was stably transfected into Chinese hamster ovary (CHO) cells. Immunoprecipitation of cellular 35S-methionine-labeled proteins with Hashimoto's serum revealed a 105-101 kilodalton doublet. In contrast, cells transfected with wild-type hTPO yielded a 112-105 kilodalton doublet. In pulse-chase experiments, CHO cells expressing the truncated hTPO protein secreted immunoprecipitable TPO into the culture medium after 4 h of chase, with levels accumulating progressively over a 24-h period. In contrast, CHO cells expressing wild-type hTPO released no immunoprecipitable TPO into the culture medium. The secreted, truncated form of hTPO appeared as a single band of lesser electrophoretic mobility, as opposed to the doublet expressed within cells. TPO enzymatic activity was present in conditioned media from CHO cells transfected with the mutated hTPO, but was absent in media from cells expressing wild-type hTPO. The stability of the mutated protein appeared similar to that of wild-type hTPO. In summary, we have generated a mutated, secreted form of hTPO that is enzymatically active and immunologically intact. Our data confirm the existence of a transmembrane domain in hTPO, and that hTPO is predominantly an enzyme with an extracellular orientation. The secreted form of hTPO has the potential for generating large amounts of soluble TPO protein for use in future structural and immunological studies.     

The propeptide domain of membrane type 1-matrix metalloproteinase acts as an intramolecular chaperone when expressed in trans with the mature sequence in COS-1 cells

It has been assumed that cleavage of the N-terminal propeptide domain of membrane type-1 matrix metalloproteinase (MT1-MMP) is required for enzyme function. We recently demonstrated that the propeptide domain of MT1-MMP is not cleaved and actually is required for function of the membrane-bound enzyme in transfected COS-1 cells (Cao, J., Drews, M., Lee, H. M., Conner, C., Bahou, W. F., and Zucker, S. (1998) J. Biol. Chem. 273, 34745-34752). In this report, we have inserted the cDNA encoding the signal and propeptide sequences of MT1-MMP (MT(1-109)) and the cDNA encoding propeptide-deleted mature MT1-MMP (MT delta pro) in expression vectors that were then transfected into matrix metalloproteinase-deficient COS-1 cells. Co-expression of both the mature sequence and the prosequence of MT1-MMP as independent polypeptides (in trans) in COS-1 cells resulted in reconstitution of MT1-MMP function in terms of facilitating (125)I-labeled tissue inhibitor of metalloproteinase 2 binding to transfected cells and subsequent activation of progelatinase A. Transfection of cells with either cDNA alone resulted in non-functional cells. These results are consistent with the propeptide sequence of MT1-MMP functioning as an intramolecular chaperone involved in protein folding and trafficking to the cell surface.     

Activation of Arabidopsis vacuolar processing enzyme by self-catalytic removal of an auto-inhibitory domain of the C-terminal propeptide

Vacuolar processing enzyme (VPE) is a cysteine proteinase responsible for the maturation of various vacuolar proteins in higher plants. To clarify the mechanism of maturation and activation of VPE, we expressed the precursors of Arabidopsis gamma VPE in insect cells. The cells accumulated a glycosylated proprotein precursor (pVPE) and an unglycosylated preproprotein precursor (ppVPE) which might be unfolded. The N-terminal sequence of pVPE revealed that ppVPE had a 22-amino-acid signal peptide to be removed co-translationally. Under acidic conditions, the 56-kDa pVPE was self-catalytically converted to a 43-kDa intermediate form (iVPE) and then to the 40-kDa mature form (mVPE). N-terminal sequencing of iVPE and mVPE showed that sequential removal of the C-terminal propeptide and N-terminal propeptide produced mVPE. Both iVPE and mVPE exhibited the activity, while pVPE exhibited no activity. These results imply that the removal of the C-terminal propeptide is essential for activating the enzyme. Further removal of the N-terminal propeptide from iVPE is not required to activate the enzyme. To demonstrate that the C-terminal propeptide functions as an inhibitor of VPE, we expressed the C-terminal propeptide and produced specific antibodies against it. We found that the C-terminal propeptide reduced the activity of VPE and that this inhibitory activity was suppressed by specific antibodies against it. Our findings suggest that the C-terminal propeptide functions as an auto-inhibitory domain that masks the catalytic site. Thus, the removal of the C-terminal propeptide of pVPE might expose the catalytic site of the enzyme.     

Degradation of human thyroperoxidase in the endoplasmic reticulum involves two different pathways depending on the folding state of the protein

Human thyroperoxidase (hTPO), a type I transmembrane glycoprotein, plays a key role in thyroid hormone synthesis. In a previous paper (Fayadat, L., Niccoli, P., Lanet, J., and Franc, J. L. (1998) Endocrinology 139, 4277-4285) we established that after the synthesis, only 15-20% of the hTPO molecules were recognized by a monoclonal antibody (mAb15) directed against a conformational structure and that only 2% were able to reach the cell surface. In the present study using pulse-chase experiments in the presence or absence of protease inhibitors followed by immunoprecipitation procedures with monoclonal antibodies recognizing unfolded or partially folded hTPO forms we show that: (i) unfolded hTPO forms are degraded by the proteasome and (ii) partially folded hTPO forms are degraded by other proteases. It was also established upon incubating endoplasmic reticulum (ER) membranes in vitro that the degradation of the partially folded hTPO was carried out by serine and cysteine integral ER membrane proteases. These data provide valuable insights into the quality control mechanisms whereby the cells get rid of misfolded or unfolded proteins. Moreover, this is the first study describing a protein degradation process involving two distinct degradation pathways (proteasome and ER cysteine/serine proteases) at the ER level, depending on the folding state of the protein.     

Cell type-dependent differences in thyroid peroxidase cell surface expression

Recently, it has been suggested that only approximately 2% of human thyroid peroxidase (hTPO(933)) reaches the surface of stably transfected (Chinese hamster ovary) cells, most being degraded intracellularly, and this might be representative of thyroid peroxidase (TPO) behavior in thyrocytes (Fayadat, L., Siffroi-Fernandez, S., Lanet, J., and Franc, J.-L. (2000) J. Biol. Chem. 275, 15948-15954). In agreement, in stably transfected Madin-Darby canine kidney clones, nonpermeabilized cells exhibit wild-type hTPO(933) immunofluorescence (apically) on <10% of that found in permeabilized cells, where an endoplasmic reticulum pattern is observed. Further, a C-terminally truncated, membrane-anchorless hTPO(848) is also retained in the endoplasmic reticulum of stably transfected Madin-Darby canine kidney cells. However, by contrast, in Chinese hamster ovary cells after transient transfection, hTPO(933) immunofluorescence is detected equally well in nonpermeabilized and permeabilized cells, indicating that a large portion of hTPO(933) is present at the cell surface; furthermore, hTPO(848) is efficiently secreted. Further, using an antiserum not cross-reacting with rat TPO, we find by immunofluorescence that in stable clones of PC Cl3 (rat) thyrocytes, considerably more ( approximately 50%) of the cells exhibit hTPO(933) at the cell surface. However, cell surface biotinylation and endoglycosidase H digestion assays appear to under-represent the extent of hTPO(933) transport, presumably because protein folding limits both Golgi carbohydrate modification and accessibility of lysines in the extracellular domain. We conclude that cell type-specific factors may facilitate stable expression of TPO at the cell surface of thyrocytes.     

Role of heme in intracellular trafficking of thyroperoxidase and involvement of H2O2 generated at the apical surface of thyroid cells in autocatalytic covalent heme binding

Thyroperoxidase (TPO) is a glycosylated hemoprotein that plays a key role in thyroid hormone synthesis. We previously showed that in CHO cells expressing human TPO (hTPO) only 2% of synthesized hTPO reaches the cell surface. Herein, we investigated the role of heme moiety insertion in the exit of hTPO from the endoplasmic reticulum. Peroxidase activity at the cell surface and cell surface expression of hTPO were decreased by approximately 30 and approximately 80%, respectively, with succinyl acetone, an inhibitor of heme biosynthesis, and were increased by 20% with holotransferrin and aminolevulinic acid, precursors of heme biosynthesis. Results were similar with holotransferrin plus aminolevulinic acid or hemin, but hemin increased cell surface activity more efficiently (+120%) relative to the control. It had been suggested (DePillis, G., Ozaki, S., Kuo, J. M., Maltby, D. A., and Ortiz de Montellano, P. R. (1997) J. Biol. Chem. 272, 8857-8960) that covalent attachment of heme to mammalian peroxidases could be an H2O2-dependent autocatalytic processing. In our study, heme associated intracellularly with hTPO, and we hypothesized that there was insufficient exposure to H2O2 in Chinese hamster ovary cells before hTPO reached the cell surface. After a 10-min incubation, 10 microM H2O2 led to a 65% increase in cell surface activity. In contrast, in thyroid cells, H2O2 was synthesized at the apical cell surface and allowed covalent attachment of heme. Two-day incubation of primocultures of thyroid cells with catalase led to a 30% decrease in TPO activity at the cell surface. In conclusion, we provide compelling evidence for an essential role of 1) heme incorporation in the intracellular trafficking of hTPO and of 2) H2O2 generated at the apical pole of thyroid cells in the autocatalytic covalent heme binding to the TPO molecule.     

Cleavage of the ADAMTS13 propeptide is not required for protease activity

ADAMTS13 belongs to the "a disintegrin and metalloprotease with thrombospondin repeats" family, and cleaves von Willebrand factor multimers into smaller forms. For several related proteases, normal folding and enzymatic latency depend on an NH2-terminal propeptide that is removed by proteolytic processing during biosynthesis. However, the ADAMTS13 propeptide is unusually short and poorly conserved, suggesting it may not perform these functions. ADAMTS13 was secreted from transfected HeLa cells with a half-time of 7 h and the rate-limiting step was exported from the endoplasmic reticulum. Deletion of the propeptide did not impair the secretion of active ADAMTS13, indicating that the propeptide is dispensable for folding. Furin was shown to be sufficient for ADAMTS13 propeptide processing in two ways. First, mutation of the furin consensus recognition site prevented propeptide cleavage in HeLa cells and resulted in secretion of pro-ADAMTS13. Second, furin-deficient LoVo cells secreted ADAMTS13 with the propeptide intact, and cotransfection with furin restored propeptide cleavage. In both cell lines, secreted pro-ADAMTS13 had normal proteolytic activity toward von Willebrand factor. In cells coexpressing both ADAMTS13 and von Willebrand factor, pro-ADAMTS13 cleaved pro-von Willebrand factor intracellularly. Therefore, the ADAMTS13 propeptide is not required for folding or secretion, and does not perform the common function of maintaining enzyme latency.     

Furin directly cleaves proMMP-2 in the trans-Golgi network resulting in a nonfunctioning proteinase

Proprotein convertases play an important role in tumorigenesis and invasiveness. Here, we report that a dibasic amino acid convertase, furin, directly cleaves proMMP-2 within the trans-Golgi network leading to an inactive form of matrix metalloproteinase-2 (MMP-2). Co-transfection of COS-1 cells with both proMMP-2 and furin cDNAs resulted in the cleavage of the N-terminal propeptide of proMMP-2. The molecular mass of cleaved MMP-2 (63 kDa), detected in both cell lysates and conditioned medium, is between the intermediate and fully activated forms of MMP-2 induced by membrane type 1-MMP. Furin-cleaved MMP-2 does not possess proteolytic activity as examined in a cell-free assay. Treatment of transfected cells with a furin inhibitor resulted in a dose-dependent inhibition of proMMP-2 cleavage; recombinant tissue inhibitor of metalloproteinase-2, which binds to the active site of membrane type 1-MMP, had no inhibitory effect. Site-directed mutagenesis of amino acids in the furin consensus recognition motif of proMMP-2(R69KPR72) prevented propeptide cleavage, thereby identifying the scissile bond and characterizing the basic amino acids required for cleavage. Other experimental observations were consistent with intracellular furin cleavage of proMMP-2 in the trans-Golgi network. The furin cleavage site in other proMMPs was examined. MMP-3, which contains the RXXR furin consensus sequence, was cleaved in furin co-transfected cells, whereas MMP-1, which lacks an RXXR consensus sequence, was not cleaved. In conclusion, we report the novel observation that furin can directly cleave the RXXR amino acid sequence in the propeptide domain of proMMP-2 leading to inactivation of the enzyme.     

General function of N-terminal propeptide on assisting protein folding and inhibiting catalytic activity based on observations with a chimeric thermolysin-like protease

Pro-aminopeptidase processing protease (PA protease) is a thermolysin-like metalloprotease produced by Aeromonas caviae T-64. The N-terminal propeptide acts as an intramolecular chaperone to assist the folding of PA protease and shows inhibitory activity toward its cognate mature enzyme. Moreover, the N-terminal propeptide strongly inhibits the autoprocessing of the C-terminal propeptide by forming a complex with the folded intermediate pro-PA protease containing the C-terminal propeptide (MC). In order to investigate the structural determinants within the N-terminal propeptide that play a role in the folding, processing, and enzyme inhibition of PA protease, we constructed a chimeric pro-PA protease by replacing the N-terminal propeptide with that of vibriolysin, a homologue of PA protease. Our results indicated that, although the N-terminal propeptide of vibriolysin shares only 36% identity with that of PA protease, it assists the refolding of MC, inhibits the folded MC to process its C-terminal propeptide, and shows a stronger inhibitory activity toward the mature PA protease than that of PA protease. These results suggest that the N-terminal propeptide domains in these thermolysin-like proteases may have similar functions, in spite of their primary sequence diversity. In addition, the conserved regions in the N-terminal propeptides of PA protease and vibriolysin may be essential for the functions of the N-terminal propeptide.     

Folding competence of N-terminally truncated forms of human procathepsin B

Besides acting as an inhibitor, the propeptide of human cathepsin B exerts an important auxiliary function as a chaperone in promoting correct protein folding. To explore the ability of N-terminally truncated forms of procathepsin B to fold into enzymatically active proteins, we produced procathepsin B variants progressively lacking N-terminal structural elements in baculovirus-infected insect cells. N-terminal truncation of the propeptide by up to 22 amino acids did not impair the production of activable procathepsin B. Secreted forms lacking the first 20, 21, or 22 amino acids spontaneously generated mature cathepsin B through autocatalytic processing, demonstrating that the first alpha-helix (Asp11-Arg20) is necessary for efficient inhibition of the enzyme by its propeptide. In contrast, proenzymes lacking the N-terminal part including the first beta-sheet (Trp24-Ala26) of the propeptide or containing an amino acid mutation directly preceding this beta-sheet were no longer properly folded. This shows that interactions between Trp24 of the propeptide and Tyr183, Tyr188, and Phe180 of the mature enzyme are important for stabilization and essential for procathepsin B folding. Thus, proenzyme forms missing more than the N-terminal 22 amino acids of the propeptide (notably truncated cathepsin B produced by the mRNA splice variant lacking exons 2 and 3, resulting in a propeptide shortened by 34 amino acids) are devoid of proteolytic activity because they cannot fold correctly. Thus, any pathophysiological involvement of truncated cathepsin B must be ascribed to properties other than proteolysis.     

The structure of the TGF-beta latency associated peptide region determines the ability of the proprotein convertase furin to cleave TGF-betas

The TGF-beta family members are generated as latent pre-pro-polypeptides. The active mature peptides are cleaved from the latent forms by cellular proteases. TGF-beta 1, for instance, is predominantly processed by a substilisin-like proprotein convertase, furin. TGF-beta 2 has a consensus cleavage site for furin and therefore has been presumed to be cleaved by furin. However, TGF-beta 2 is often secreted as the latent form, which appears to be inconsistent with its postulated sensitivity to furin. We report here that both the regular (short) form of TGF-beta2 and its spliced variant with an additional exon (long form) are insensitive to furin. NIH 3T3 and CHO cells were transfected with expression vectors containing the short or long form of TGF-beta 2 or a chimeric TGF-beta consisting of the TGF-beta1 LAP region, the TGF-beta 2 cleavage site and the TGF-beta 2 mature peptide. The constructs included a c-myc epitope tag in the N-terminal region of the mature peptide. The TGF-betas produced by the transfected cells were analyzed with Western blots and immunocytochemistry. The intracellular proteins harvested from these cells were incubated with furin. Furin only inefficiently cleaved both the long and short forms of TGF-beta 2, but efficiently processed the chimeric TGF-beta. This indicates that the insensitivity of both forms of TGF-beta 2 to furin is a consequence of the tertiary structure of their LAP regions rather than their cleavage site. This differential processing of TGF-beta1 and -beta 2 may be part of the mechanism that generates isoform-specific functions of the TGF-betas.     

Regulation of the autoactivation of human 72-kDa progelatinase by tissue inhibitor of metalloproteinases-2

To study the activation of human 72-kDa gelatinase, and its relation to tissue inhibitor of metalloproteinases 2 (TIMP-2), we purified human 72-kDa progelatinase both as a complex with TIMP-2 and as a free proteinase. Activation of progelatinase-TIMP-2 complexes with 4-aminophenylmercuric acetate yielded gelatinolytically active enzyme migrating at 62 kDa. TIMP-2 remained bound to the active enzyme. Removal of TIMP-2 from progelatinase by reverse-phase high performance liquid chromatography in the presence of trifluoroacetic acid, followed by complete dialysis in neutral pH buffer, resulted in multiple fragments. These fragments were formed as a result of the cleavage of 72-kDa progelatinase at several locations. Cleavage at the amino terminus was restricted to the removal of the propeptide, except in the case of degradation leading to inactive fragments. Two active species autocatalytically evolved upon removal of TIMP-2 from progelatinase. The 62 kDa-activated gelatinase lacked the amino-terminal propeptide, which is known to be removed upon treatment with 4-aminophenylmercuric acetate. In addition, an active 42.5-kDa fragment lacking both the propeptide and a portion of the carboxyl terminus was formed. This low-molecular-weight active form of 72-kDa progelatinase retained its ability to bind and degrade gelatin. Self-activation and degradation of 72-kDa progelatinase can be prevented by agents that inhibit metalloproteinases, including 1,10-phenanthroline. Evidence presented here suggests that TIMP-2 binds to a stabilization site that is independent of the active site. This stabilization site does not bind TIMP-1 (TIMP). Occupation of this site by TIMP-2 prevents autocatalytic activation and degradation but does not prevent gelatinolysis by the enzyme-inhibitor complex.     

Activation of the Kexin from Schizosaccharomyces pombe Requires Internal Cleavage of Its Initially Cleaved Prosequence

Members of the kexin family of processing enzymes are responsible for the cleavage of many proproteins during their transport through the secretory pathway. The enzymes themselves are made as inactive precursors, and we investigated the activation process by studying the maturation of Krp1, a kexin from the fission yeast Schizosaccharomyces pombe. Using a cell-free translation-translocation system prepared from Xenopus eggs, we found that Krp1 is made as a preproprotein that loses the presequence during translocation into the endoplasmic reticulum. The prosequence is also rapidly cleaved in a reaction that is autocatalytic and probably intramolecular and is inhibited by disruption of the P domain. Prosequence cleavage normally occurs at Arg-Tyr-Lys-Arg102↓ (primary cleavage site) but can occur at Lys-Arg82 (internal cleavage site) and/or Trp-Arg99 when the basic residues are removed from the primary site. Cleavage of the prosequence is necessary but not sufficient for activation, and Krp1 is initially unable to process substrates presented in trans. Full activation is achieved after further incubation in the extract and is coincident with the addition of O-linked sugars. O glycosylation is not, however, essential for activity, and the crucial event appears to be cleavage of the initially cleaved prosequence at the internal site. Our results are consistent with a model in which the cleaved prosequence remains noncovalently associated with the catalytic domain and acts as an autoinhibitor of the enzyme. Inhibition is then relieved by a second (internal) cleavage of the inhibitory prosequence. Further support for this model is provided by our finding that overexpression of a Krp1 prosequence lacking a cleavable internal site dramatically reduced the growth rate of otherwise wild-type S. pombe cells, an effect that was not seen after overexpression of the normal, internally cleavable, prosequence or prosequences that lack the Lys-Arg102 residues.     

The propeptide is not required to produce catalytically active neutral protease from Bacillus stearothermophilus

The thermolysin-like neutral protease from Bacillus stearothermophilus (TLP-ste) is usually produced extracellularly in Bacillus subtilis, where it is expressed as preproenzyme and subsequently processed in an autocatalytic, intramolecular process. To create the basis for the production of inactive mutants of TLP-ste, which cannot be processed in B. subtilis, we studied the expression of TLP-ste and its propeptide in cis and in trans in Escherichia coli. In contrast to thermolysin, subtilisin and alpha-lytic protease, which could be obtained only in the presence of the corresponding propeptides, TLP-ste could be produced as an active mature enzyme in E. coli in the absence of its prosequence. Surprisingly, however, a much more effective access to active mature protease was found when TLP-ste (devoid of its prosequence) was expressed as protein with an N-terminal His6 tag which accumulated in the form of inclusion bodies. Completely unexpected, the protein could be renatured from the inclusion bodies after solubilization in guanidine hydrochloride solutions in high yields. Purification to homogeneity was possible by affinity chromatography on Bacitracin silica as well as by immobilized metal ion affinity chromatography. By addition of separately expressed propeptide to the renaturation mixture yields of renaturation could not be increased significantly, confirming that the propeptide is not essential for proper folding of the enzyme or its stabilization during the folding process. Also in vivo, the expression levels of active mature TLP-ste in Escherichia coli did not significantly differ when the mature sequence was expressed alone or coexpressed with the prosequence in cis or in trans.     

Degradation of T-cell receptor chains in the endoplasmic reticulum is inhibited by inhibitors of cysteine proteases.

The endoplasmic reticulum, or an organelle closely associated with it, contains proteases that can be used to remove partially assembled or improperly folded proteins. Very little is known at present about the types of protease that degrade these proteins. The beta chain and cluster of differentiation (CD)3 delta subunit of the human T-cell antigen receptor (TCR) are degraded shortly after synthesis. In this study Chinese hamster ovary (CHO) cells transfected with either beta or delta were incubated with a panel of protease inhibitors, and the rates of degradation of the transfected proteins were followed using chain-specific enzyme-linked immunosorbent assays (ELISAs). Of the protease inhibitors tested, degradation of both chains was highly sensitive to sulfhydryl reagents and peptidyl inhibitors of cysteine proteases. Concentrations of inhibitors that produced near complete inhibition of degradation in the endoplasmic reticulum did not cause gross changes in cellular ATP levels nor did they significantly slow constitutive secretion from CHO cells. The inhibitors did not affect the ability of CHO cells to synthesize and assemble disulphide-linked TCR zeta dimers. We conclude that the protease inhibitors were not toxic to cells and did not affect the biosynthetic activity of the endoplasmic reticulum. Furthermore, they did not alter the ability of the endoplasmic reticulum to deliver its content to the Golgi apparatus. Taken together, these results suggest that the cysteine protease inhibitors slow degradation in the endoplasmic reticulum through an action on cysteine proteases. The results imply that the endoplasmic reticulum contains cysteine proteases that can be used to remove retained proteins.     

The N-terminal prepeptide is required for the production of spore cortex-lytic enzyme from its inactive precursor during germination of Clostridium perfringens S40 spores

A spore cortex-lytic enzyme of Clostridium perfringens S40 is synthesized during sporulation as a precursor consisting of four domains. After cleavage of an N-terminal preregion and a C-terminal proregion, inactive proenzyme (termed C35) is converted to active enzyme by processing of an N-terminal prosequence with germination-specific protease (GSP) during germination. The present results demonstrated that the cleaved N-terminal prepeptide remained associated with C35. After the isolated complex was denatured and dissociated in 6 M urea solution, removal of urea regenerated a prepeptide-C35 complex which produces active enzyme when incubated with GSP. However, isolated C35 alone could not be activated by GSP. The prepeptide-C35 complex was more heat stable than active enzyme. Thus, non-covalent attachment of the prepeptide to C35 is required to assist correct folding of C35 and to stabilize its conformation, suggesting that the prepeptide functions as an intramolecular chaperone. Recombinant proteins, which have prepeptide covalently bonded to C35, were processed by GSP as well as the in vivo prepeptide-C35 complex, and the full length of the N-terminal presequence was needed to fulfil its role. Although the C-terminal prosequence is present as an independent domain which is not involved in the activation process of the enzyme, it appears that the N-terminal prosequence contributes to the regulation of enzyme activity as an inhibitor of the enzyme.     

The role of the N-terminal propeptide of the pro-aminopeptidase processing protease: refolding,processing, and enzyme inhibition

Pro-aminopeptidase processing protease (PA protease) is an extracellular zinc metalloprotease produced by Aeromonas caviae T-64 and it is classified as M04.016 according to the MEROPS database. The precursor of PA protease consists of four regions; a signal peptide, an N-terminal propeptide, a C-terminal propeptide, and the mature PA protease. The in vitro refolding of the intermediate pro-PA protease containing the C-terminal propeptide (MC) was investigated in the presence and absence of the N-terminal propeptide. The results indicate that the noncovalently linked N-terminal propeptide is able to assist in the refolding of MC. In the absence of the N-terminal propeptide, MC is trapped into a folding competent state that is converted into the active form by the addition of the N-terminal propeptide. Moreover, the N-terminal propeptide was found to form a complex with the folded MC and inhibit further processing of MC into the mature PA protease. Inhibitory activity of the purified N-terminal propeptide toward mature PA protease was also observed, and the mode of this inhibition was determined to be a mixed, noncompetitive inhibition with an associated allosteric effect.     

Efficient production of active Vibrio proteolyticus aminopeptidase in Escherichia coli by co-expression with engineered vibriolysin

The Vibrio proteolyticus aminopeptidase is synthesized as a preproprotein and then converted into an active enzyme by cleavage of the N-terminal propeptide. In recombinant Escherichia coli, however, the aminopeptidase is not processed correctly and the less-active form that has the N-terminal propeptide accumulates in the culture medium. Recently, we isolated a novel vibriolysin that was expressed as an active form in E. coli by random mutagenesis; this enzyme shows potential as a candidate enzyme for the processing of aminopeptidase. The E. coli cells were engineered to co-express the novel vibriolysin along with aminopeptidase. Co-expression of vibriolysin resulted in an approximately 13-fold increase in aminopeptidase activity, and a further increase was observed in the form lacking its C-terminal propeptide. The active aminopeptidase was purified from the culture supernatant including the recombinant vibriolysin by heat treatment and ion exchange and hydroxyapatite chromatography with high purity and 35% recovery rate. This purified aminopeptidase effectively converted methionyl-human growth hormone (Met-hGH) to hGH. Thus, this co-expression system provides an efficient method for producing active recombinant V. proteolyticus aminopeptidase.     

A covalent oxidoreductase intermediate in propeptide-dependent von Willebrand factor multimerization

The assembly of von Willebrand factor multimers in the Golgi apparatus requires D1D2 domains of the von Willebrand factor propeptide, which may act as an oxidoreductase to promote disulfide bond formation or rearrangement between two D3 domains in the mature subunit. This mechanism predicts that the propeptide should form a transient intrachain disulfide bond with the D3 domain before multimerization. Such an intermediate was detected using truncated subunits that simplify the analysis of the multimerization process. When only the D1D2D'D3 region of von Willebrand factor was expressed in baby hamster kidney cells, the propeptide and D'D3 formed an intrachain disulfide-linked species in the endoplasmic reticulum that could be identified by two-dimensional gel electrophoresis after cleavage with thrombin or furin. This intermediate rearranged in the Golgi to form free propeptide and D'D3 dimers that were secreted. A similar intracellular disulfide-linked species was identified in cells expressing the propeptide and D'D3 as separate proteins and in cells expressing full-length von Willebrand factor. These results support a model in which the propeptide acts as an oxidoreductase to promote von Willebrand factor multimerization in the Golgi apparatus.     

Biosynthesis and post-translational processing of the precursor to brain-derived neurotrophic factor

We examined the biosynthesis and post-translational processing of the brain-derived neurotrophic factor precursor (pro-BDNF) in cells infected with a pro-BDNF-encoding vaccinia virus. Metabolic labeling, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis reveal that pro-BDNF is generated as a 32-kDa precursor that is N-glycosylated and glycosulfated on a site, within the pro-domain. Some pro-BDNF is released extracellularly and is biologically active as demonstrated by its ability to mediate TrkB phosphorylation. The precursor undergoes N-terminal cleavage within the trans-Golgi network and/or immature secretory vesicles to generate mature BDNF (14 kDa). Small amounts of a 28-kDa protein that is immunoprecipitated with BDNF antibodies is also evident. This protein is generated in the endoplasmic reticulum through N-terminal cleavage of pro-BDNF at the Arg-Gly-Leu-Thr(57)- downward arrow-Ser-Leu site. Cleavage is abolished when Arg(54) is changed to Ala (R54A) by in vitro mutagenesis. Blocking generation of 28-kDa BDNF has no effect on the level of mature BDNF and blocking generation of mature BDNF with alpha(1)-PDX, an inhibitor of furin-like enzymes, does not lead to accumulation of the 28-kDa form. These data suggest that 28-kDa pro-BDNF is not an obligatory intermediate in the formation of the 14-kDa form in the constitutive secretory pathway.