The propeptide of von Willebrand factor independently mediates the assembly of von Willebrand multimers

The biosynthesis of von Willebrand Factor (vWF) by vascular endothelial cells involves a complex series of processing steps that includes proteolytic cleavage of a 741-residue propeptide and the assembly of disulfide-linked multimers. Using a model system in which experimentally altered vWF cDNAs are expressed in COS-1 cells, we have shown that the vWF propeptide contains determinants that govern the assembly of vWF multimers. Furthermore, the role of the propeptide (in the assembly process) does not require it to be a contiguous part of the pro-vWF primary structure, since independently expressed propeptide was shown to promote the assembly of mature vWF subunits into multimers. Pulse-chase experiments indicated that the independently expressed propeptide formed a transient association with the mature vWF subunit inside the cell. Thus, it appears that the vWF propeptide segment can act in "trans" to direct the assembly of disulfide-linked vWF multimers.     

The role of von Willebrand factor multimers and propeptide cleavage in binding and stabilization of factor VIII

von Willebrand factor (vWF) is a multimeric glycoprotein that promotes platelet aggregation and stabilizes coagulation factor VIII in the plasma. vWF is also required for the stable accumulation of recombinant factor VIII secreted from cells in a heterologous expression system. In this report, we show that vWF can promote the in vitro reconstitution of factor VIII activity from dissociated heavy and light chains of factor VIII, suggesting that vWF may act to promote stable assembly of factor VIII subunits at the site of secretion. The structural requirements for vWF propeptide cleavage and for vWF multimerization in its binding and stabilization of factor VIII was examined using specifically altered recombinant vWF. The mutant vWF molecules were also assayed for their function in ristocetin-induced platelet agglutination mediated through the platelet receptor GPIb. Deletion of the vWF propeptide produced a dimeric vWF molecule that failed to mediate platelet agglutination, suggesting that multimerization is required for vWF to attain functional GPIb binding. This mature dimeric form of vWF, however, was fully capable of binding to and supporting stable secretion of factor VIII. A vWF mutant with an altered propeptide cleavage site formed large multimers of uncleaved pro-vWF that functioned in platelet agglutination. However, this noncleavage mutant neither bound to or supported stable accumulation of factor VIII. Analysis of the vWF propeptide, expressed independently, demonstrated that it could not bind factor VIII or stabilize its secretion. These results show that the dimeric mature vWF subunit is sufficient to bind and stabilize factor VIII and that the presence of uncleaved vWF propeptide inhibits both factor VIII binding and stabilization.     

Proteolytic cleavage of the precursor of von Willebrand factor is not essential for multimer formation

Monkey kidney cells (COS-1), transfected with full-length human von Willebrand factor (vWF) cDNA encoding the precursor of vWF (pro-vWF), mimic the characteristics of the biosynthesis and of the constitutive secretory pathway, displayed by cultured vascular endothelial cells. Such heterologous transfected cells are able to cleave pro-vWF, generating the propolypeptide and mature vWF, and to assemble pro-vWF dimers into a series of multimers, similarly to endothelial cells. Evidence is presented showing that proteolytic processing of pro-vWF by COS-1 cells occurs at the peptide bond between arginine and serine in the sequence Lys762-Arg763-Ser764, identical to endothelial cell-associated proteolysis. This conclusion stems from the observation that substitution of Arg763 by a glycine residue completely abolishes proteolytic processing. As a result, transfection of COS-1 with the mutant vWF-Gly763 cDNA does not significantly affect the multimeric organization of secreted vWF molecules. Consequently, we conclude that proteolytic processing of pro-vWF is not required for multimer formation. Pulse-chase labeling of COS-1 cells transfected with full-length vWF cDNA reveals pro-vWF exclusively in cell lysates, whereas both pro-vWF and mature vWF are encountered in the conditioned medium. These observations indicate that proteolytic processing of pro-vWF is a "late" event during intracellular routing of these molecules or may occur extracellularly.     

Domains involved in multimer assembly of von willebrand factor (vWF): multimerization is independent of dimerization.

The precursor protein of von Willebrand factor (pro-vWF) consist of four repeated domains, denoted D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2. The domains D1 and D2 constitute the amino-terminal pro-polypeptide and the remaining domains mature vWF, generated upon proteolytic processing. We have shown previously that the pro-polypeptide of pro-vWF is obligatory for assembly of pro-vWF dimers into multimers, a process vital for efficient adhesion of platelets to an injured vessel wall. Here, we have employed full length vWF cDNA to construct a series of deletion mutants, based on the homology between the various domains. Specifically, the domains D', D3 or both were deleted and the multimeric pattern of the mutant vWF proteins was analysed after transient expression in COS-1 cells. It is demonstrated that in addition to the pro-polypeptide, both the D' and the D3 domain are required for multimer assembly. Furthermore, by analysing a construct containing only the domains D' and D3 next to the pro-polypeptide it is shown that this is the only part of the vWF protein involved in multimer assembly. Since, the formation of pro-vWF dimers relies on the carboxy-terminal area of mature vWF, it is concluded that multimer assembly is a process independent of dimerization.     

Independent assembly and secretion of a dimeric adhesive domain of von Willebrand factor containing the glycoprotein Ib-binding site

von Willebrand factor (vWF) is a multimeric glycoprotein that supports platelet adhesion on thrombogenic surfaces as part of the normal hemostatic response to vascular injury. We have employed a domain-specific expression strategy to analyze the biosynthetic processing steps and minimum structural requirements for assembly of the platelet receptor glycoprotein Ib-binding domain of vWF. A chimeric cDNA that codes for the vWF signal peptide and a segment of vWF internal primary sequence, residues 441-730, directs the secretion of a functional vWF fragment from mammalian cells. The recombinant molecule intrinsically assembles through intermolecular disulfide bond formation into a dimeric adhesive domain without contributions from other regions of vWF, including propeptide, previously indicated as essential for vWF multimer assembly. Prevention of N-linked glycosylation on the recombinant domain does not impair dimer formation or the ability to support platelet aggregation. These results identify a minimum structural element for vWF subunit assembly and provide new insights into the processing steps to produce vWF multimers and adhesive domains.     

A furin-like convertase mediates propeptide cleavage of BACE, the Alzheimer's beta -secretase

The novel transmembrane aspartic protease BACE (for Beta-site APP Cleaving Enzyme) is the beta-secretase that cleaves amyloid precursor protein to initiate beta-amyloid formation. As such, BACE is a prime therapeutic target for the treatment of Alzheimer's disease. BACE, like other aspartic proteases, has a propeptide domain that is removed to form the mature enzyme. BACE propeptide cleavage occurs at the sequence RLPR downward arrowE, a potential furin recognition motif. Here, we explore the role of furin in BACE propeptide domain processing. BACE propeptide cleavage in cells does not appear to be autocatalytic, since an inactive D93A mutant of BACE is still cleaved appropriately. BACE and furin co-localize within the Golgi apparatus, and propeptide cleavage is inhibited by brefeldin A and monensin, drugs that disrupt trafficking through the Golgi. Treatment of cells with the calcium ionophore, leading to inhibition of calcium-dependent proteases including furin, or transfection with the alpha(1)-antitrypsin variant alpha(1)-PDX, a potent furin inhibitor, dramatically reduces cleavage of the BACE propeptide. Moreover, the BACE propeptide is not processed in the furin-deficient LoVo cell line; however, processing is restored upon furin transfection. Finally, in vitro digestion of recombinant soluble BACE with recombinant furin results in complete cleavage only at the established E46 site. Taken together, our results strongly suggest that furin, or a furin-like proprotein convertase, is responsible for cleaving the BACE propeptide domain to form the mature enzyme.     

Biogenesis of von Willebrand factor-containing organelles in heterologous transfected CV-1 cells.

Von Willebrand factor (vWF) is a multimeric protein involved in the adhesion of platelets to an injured vessel wall. vWF is synthesized by the endothelial cell and the megakaryocyte as a precursor protein (pro-vWF) that consists of four repeated domains, denoted D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2. Previously, we have defined the domains on the pro-vWF molecule involved in dimerization as well as the domains involved in multimer assembly of vWF dimers. In the endothelial cell, part of the vWF multimers is stored in specialized organelles, the Weibel-Palade bodies. By using immunoelectron microscopy, we demonstrate that upon expression of full-length vWF cDNA, vWF-containing organelles are encountered in monkey kidney CV-1 cells that are morphologically similar to the endothelial-specific Weibel-Palade bodies. Expression in CV-1 cells of a series of vWF cDNA deletion mutants, lacking one or more domains, revealed that only those vWF mutant proteins that are able to assemble into multimers are encountered in dense-cored vesicles. Our data show that this process is independent of a particular domain on vWF and indicate that a 'condensed', multimeric vWF is required for targeting to the Weibel-Palade body.     

Assembly and routing of von Willebrand factor variants: the requirements for disulfide-linked dimerization reside within the carboxy-terminal 151 amino acids

The precursor protein of von Willebrand factor (pro-vWF) consists of four different repeated domains, denoted D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2, followed by a carboxy-terminal region of 151 amino acids without obvious internal homology. Previously, we have shown the requirement of the domains D1, D2, D', and D3 of pro-vWF in the assembly of pro-vWF dimers into multimers. Here, we define the domains of vWF involved in dimerization, using deletion mutants of full-length vWF cDNA transiently expressed in monkey kidney COS-1 cells. It is shown that only the carboxy-terminal 151 amino acid residues of vWF are required for dimerization. In addition, by analyzing a construct, encoding only the carboxy-terminal 151 amino acids of vWF, we find that the formation of dimers is an event independent of other domains present on pro-vWF, such as the domains C1 and C2 previously suggested to be involved in dimerization. Furthermore, it is shown that a deletion mutant of vWF, lacking the carboxy-terminal 151 amino acid residues and thus unable to dimerize, is proteolytically degraded in the ER. In contrast, a mutant protein, composed only of the carboxy-terminal 151 amino acids of vWF, and able to dimerize, is transported from the ER in a similar fashion as wild-type vWF. The role of the ER in the assembly of vWF is discussed with regard to the data presented in this paper on the intracellular fate of several vWF mutant proteins.     

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.     

Propeptide cleavage conditions sortilin/neurotensin receptor-3 for ligand binding.

We recently reported the isolation and sequencing of sortilin, a new putative sorting receptor that binds receptor-associated protein (RAP). The luminal N-terminus of sortilin comprises a consensus sequence for cleavage by furin, R41WRR44, which precedes a truncation originally found in sortilin isolated from human brain. We now show that the truncation results from cellular processing. Sortilin is synthesized as a proform which, in late Golgi compartments, is converted to the mature receptor by furin-mediated cleavage of a 44 residue N-terminal propeptide. We further demonstrate that the propeptide exhibits pH-dependent high affinity binding to fully processed sortilin, that the binding is competed for by RAP and the newly discovered sortilin ligand neurotensin, and that prevention of propeptide cleavage essentially prevents binding of RAP and neurotensin. The findings evidence that the propeptide sterically hinders ligands from gaining access to overlapping binding sites in prosortilin, and that cleavage and release of the propeptide preconditions sortilin for full functional activity. Although proteolytic processing is involved in the maturation of several receptors, the described exposure of previously concealed ligand-binding sites after furin-mediated cleavage of propeptide represents a novel mechanism in receptor activation.     

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.     

Processing of pro-thrombostasin by a recombinant subtilisin-like proprotein convertase derived from the salivary glands of horn flies (Haematobia irritans)

Thrombostasin (TS) is a thrombin inhibitor found in the salivary glands of horn flies (Haematobia irritans). It is produced as an inactive form with a 76-amino acid propeptide in the N-terminus preceding the mature TS. A minimal recognition sequence by subtilisin-like proprotein convertases, Arg-Xaa-Xaa-Arg, is localized C-terminal to the propeptide. This study demonstrated that a gene cloned from the salivary glands of the horn fly encodes a new convertase, subsequently named horn fly proprotein convertase (HFPC), and that the recombinant HFPC expressed in insect HighFive cell culture specifically cleaves recombinant pro-thrombostasin, produced in E. coli, at the expected site. The relative cleavage efficiency of rHFPC was compared with that of recombinant human furin, a commercially available proprotein convertase. The result indicated that this newly identified proprotein convertase is of importance for the proteolytic maturation of thrombostasin, a protein secreted in horn fly saliva and used by the insect to counteract its host's haemostatic response.     

Extracellular production of active vibriolysin engineered by random mutagenesis in Escherichia coli

Vibriolysin, an extracellular protease of Vibrio proteolyticus, is synthesized as a preproenzyme. The N-terminal propeptide functions as an intramolecular chaperone and an inhibitor of the mature enzyme. Extracellular production of recombinant vibriolysin has been achieved in Bacillus subtilis, but not in Escherichia coli, which is widely used as a host for the production of recombinant proteins. Vibriolysin is expressed as an inactive form in E. coli possibly due to the inhibitory effect of the N-terminal propeptide. In this study, we isolated the novel vibriolysin engineered by in vivo random mutagenesis, which is expressed as active mature vibriolysin in E. coli. The Western blot analysis showed that the N-terminal propeptide of the engineered enzyme was processed and degraded, confirming that the propeptide inhibits the mature enzyme. Two mutations located within the engineered vibriolysin resulted in the substitution of stop codon for Trp at position 11 in the signal peptide and of Val for Ala at position 183 in the N-terminal propeptide (where position 1 is defined as the first methionine). It was found that the individual mutations are related to the enzyme activity. The novel vibriolysin was extracellularly overproduced in BL21(DE3) and purified from the culture supernatant by ion-exchange chromatography followed by hydrophobic-interaction chromatography, resulting in an overall yield of 2.2 mg/L of purified protein. This suggests that the novel engineered vibriolysin is useful for overproduction in an E. coli expression system.     

Processing of pulmonary surfactant protein B by napsin and cathepsin H

Surfactant protein B (SP-B) is an essential constituent of pulmonary surfactant. SP-B is synthesized in alveolar type II cells as a preproprotein and processed to the mature peptide by the cleavage of NH2- and COOH-terminal peptides. An aspartyl protease has been suggested to cleave the NH2-terminal propeptide resulting in a 25-kDa intermediate. Napsin, an aspartyl protease expressed in alveolar type II cells, was detected in fetal lung homogenates as early as day 16 of gestation, 1 day before the onset of SP-B expression and processing. Napsin was localized to multivesicular bodies, the site of SP-B proprotein processing in type II cells. Incubation of SP-B proprotein from type II cells with a crude membrane extract from napsin-transfected cells resulted in enhanced levels of a 25-kDa intermediate. Purified napsin cleaved a recombinant SP-B/EGFP fusion protein within the NH2-terminal propeptide between Leu178 and Pro179, 22 amino acids upstream of the NH2 terminus of mature SP-B. Cathepsin H, a cysteine protease also implicated in pro-SP-B processing, cleaved SP-B/EGFP fusion protein 13 amino acids upstream of the NH2 terminus of mature SP-B. Napsin did not cleave the COOH-terminal peptide, whereas cathepsin H cleaved the boundary between mature SP-B and the COOH-terminal peptide and at several other sites within the COOH-terminal peptide. Knockdown of napsin by small interfering RNA resulted in decreased levels of mature SP-B and mature SP-C in type II cells. These results suggest that napsin, cathepsin H, and at least one other enzyme are involved in maturation of the biologically active SP-B peptide.     

Alternative cleavage of the cachectin/tumor necrosis factor propeptide results in a larger, inactive form of secreted protein

Cachectin/tumor necrosis factor is initially synthesized as a 26 kilodalton prohormone. This molecule is 76 (human) or 79 (mouse) amino acids longer than the mature protein, as a result of additional residues present at the amino terminus. A short hydrophobic stretch of amino acids serves to anchor the prohormone in lipid bilayers; the mature protein, as well as a partially processed form of the hormone, are secreted after cleavage of the propeptide. We have analyzed the cleavage of the murine propeptide as it occurs in RAW 264.7 cells and now report that scission occurs at a site within the propeptide fragment, 10 residues before the amino-terminal leucine of the mature protein. This incompletely processed form of the molecule also begins with a leucine residue. Although secreted as a soluble product, it is biologically inactive in the L-929 cell cytotoxicity assay.     

Expression of variant von Willebrand factor (vWF) cDNA in heterologous cells: requirement of the pro-polypeptide in vWF multimer formation.

Von Willebrand factor (vWF) is a multimeric plasma glycoprotein synthesized by vascular endothelial cells as a pre-pro-polypeptide with a highly repetitive domain structure, symbolized by the formula: (H)-D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-(OH) A heterologous expression system for the synthesis of recombinant vWF protein was developed, consisting of a monkey kidney cell line (COS-1), transfected with full-length vWF cDNA. This system was shown to mimic the constitutive secretory pathway of vWF in endothelial cells, since dimerization and multimerization occur similarly. To determine whether the pro-polypeptide, composed of the domains D1 and D2, is involved in vWF multimerization, a vWF cDNA was constructed that lacked the coding sequence for the pro-polypeptide. The mutant vWF protein, expressed by COS-1 cells transfected with this cDNA, did not assemble beyond the dimer stage. From this observation, we conclude that (i) dimerization does not involve the pro-polypeptide of pro-vWF and (ii) the presence of the pro-polypeptide, as part of pro-vWF, is obligatory for multimerization. It is argued that the interactions, required for interchain binding, are mediated by the D domains.     

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.     

Autocatalytic cleavage of human gamma-glutamyl transpeptidase is highly dependent on N-glycosylation at asparagine 95

γ-Glutamyl transpeptidase (GGT) is a heterodimeric membrane enzyme that catalyzes the cleavage of extracellular glutathione and other γ-glutamyl-containing compounds. GGT is synthesized as a single polypeptide (propeptide) that undergoes autocatalytic cleavage, which results in the formation of the large and small subunits that compose the mature enzyme. GGT is extensively N-glycosylated, yet the functional consequences of this modification are unclear. We investigated the effect of N-glycosylation on the kinetic behavior, stability, and functional maturation of GGT. Using site-directed mutagenesis, we confirmed that all seven N-glycosylation sites on human GGT are modified by N-glycans. Comparative enzyme kinetic analyses revealed that single substitutions are functionally tolerated, although the N95Q mutation resulted in a marked decrease in the cleavage efficiency of the propeptide. However, each of the single site mutants exhibited decreased thermal stability relative to wild-type GGT. Combined mutagenesis of all N-glycosylation sites resulted in the accumulation of the inactive propeptide form of the enzyme. Use of N-glycosylation inhibitors demonstrated that binding of the core N-glycans, not their subsequent processing, is the critical glycosylation event governing the autocleavage of GGT. Although N-glycosylation is necessary for maturation of the propeptide, enzymatic deglycosylation of the mature wild-type GGT does not substantially impact either the kinetic behavior or thermal stability of the fully processed human enzyme. These findings are the first to establish that co-translational N-glycosylation of human GGT is required for the proper folding and subsequent cleavage of the nascent propeptide, although retention of these N-glycans is not necessary for maintaining either the function or structural stability of the mature enzyme.     

Mutational analysis of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase propeptide processing

Glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis is a member of an N-terminal nucleophile hydrolase enzyme superfamily, several of which undergo autocatalytic propeptide processing to generate the mature active enzyme. A series of mutations was analyzed to determine whether amino acid residues required for catalysis are also used for propeptide processing. Propeptide cleavage was strongly inhibited by replacement of the cysteine nucleophile and two residues of an oxyanion hole that are required for glutaminase function. However, significant propeptide processing was retained in a deletion mutant with multiple defects in catalysis that was devoid of enzyme activity. Intermolecular processing of noncleaved mutant enzyme subunits by active wild-type enzyme subunits was not detected in hetero-oligomers obtained from a coexpression experiment. While direct in vitro evidence for autocatalytic propeptide cleavage was not obtained, the results indicate that some but not all of the amino acid residues that have a role in catalysis are also needed for propeptide processing.