Identification of a functional proprotein convertase cleavage site in microfibril-associated glycoprotein 2

Microfibril-associated glycoprotein 2 (MAGP2) is a secreted protein associated with multiple cellular activities including the organization of elastic fibers in the extracellular matrix (ECM), angiogenesis, as well as regulating Notch and integrin signaling. Importantly, increases in MAGP2 positively correlate with poor prognosis for some ovarian cancers. It has been assumed that full-length MAGP2 is responsible for all reported effects; however, here we show MAGP2 is a substrate for the proprotein convertase (PC) family of endoproteases. Proteolytic processing of MAGP2 by PC cleavage could serve to regulate secretion and thus, activity and function as reported for other extracellular and cell-surface proteins. In support of this idea, MAGP2 contains an evolutionarily conserved PC consensus cleavage site, and amino acid sequencing of a newly identified MAGP2 C-terminal cleavage product confirmed functional PC cleavage. Additionally, mutagenesis of the MAGP2 PC consensus cleavage site or treatment with PC inhibitors prevented MAGP2 proteolytic processing. Finally, both cleaved and uncleaved MAGP2 were detected extracellularly and MAGP2 secretion appeared independent of PC cleavage, suggesting that PC processing occurs mainly outside the cell. Our characterization of alternative forms of MAGP2 present in the extracellular space not only enhances diversity of this ECM protein but also provides a previously unrecognized molecular mechanism for regulation of MAGP2 biological activity.     

Hydrogen peroxide cleavage of the prion protein generates a fragment able to initiate polymerisation of full length prion protein

The prion protein is central to the disease pathogenesis of a variety of neurodegenerative diseases such as CJD. The protein is only able to initiate the disease process following post-translational modification. The main characteristic of this change is the ability of this altered isoform to polymerise. We wish to determine if altered cleavage of the protein could generate a protein fragment able to initiate polymerisation. During normal metabolic breakdown the protein is initially cleaved at a single site at around amino acid residue 111/112 in the mouse sequence. A second site before amino acid residue 90 has been postulated as an alternative cleavage point. We have provided evidence that hydrogen peroxide as low as 50 microM in the presence of copper, iron or manganese (but not nickel, magnesium or zinc) can cleave the recombinant protein near this site and requires a GXXH motif in the protein sequence. This reaction results in the production of 6 and 19 kDa fragments of the protein. This cleavage pattern occurs in prion proteins from different species (mouse, chicken and turtle) and is enhanced by modification of the octameric repeat region. The 19 kDa fragment produced by this reaction is protease sensitive. This fragment in a pure form caused the polymerisation of wild-type prion protein by a seeding mechanism. Therefore our results provide a possible mechanism by which altered cleavage of the prion protein could result in the kind of protein polymerisation associated with prion diseases.     

Immunogenicity of a bovine rotavirus glycoprotein fragment.

Previous experiments demonstrated that an antigenic site responsible for virus neutralization and cell attachment was located on a 14,000-molecular-weight fragment of the major bovine rotavirus (BRV) glycoprotein (M. Sabara, J. E. Gilchrist, G. R. Hudson, and L. A. Babiuk, J. Virol. 53:58-66, 1985). However, it was necessary to investigate whether this fragment also had the ability to induce the production of neutralizing antibodies. Upon immunization of mice, the bovine serum albumin-conjugated 14,000-molecular-weight fragment, the unconjugated 14,000-molecular-weight fragment, and the native glycoprotein all induced a similar neutralizing antibody response, albeit to a lesser extent than did the infectious, whole virus. In addition, immuno-blot enzyme-linked immunosorbent assay analysis of the reactivity of anti-peptide serum versus anti-glycoprotein serum with the glycoprotein was very comparable. These results suggest that the 14,000-molecular-weight fragment may represent not only a biologically active region but also an immunodominant area of the glycoprotein.     

Direct cleavage of a DNA fragment by a bleomycin-oligonucleotide derivative

Bicomycin A₁ oligonucleotide derivative was used for direct cleavage of a DNA target. In the presence of Fe²⁺ ions and 2-mercaptoethanol, Blm-R-pd(CCAAACA) (I) damaged the target, pd(TGTTTGGCGAAGGA), with the yield of 80%, without affecting its own oligonucleotide tail. The sites of the cleavage were T^?-T^? and G^?-G^?. Unbound bleomycin A₅, damaged the G⁶-G⁷-G⁸ site. Reagent I formed more stable complementary complexes with the target than parent oligonucleotide (ΔT_m=11°C)     

Proprotein convertase activity contributes to the processing of the Alzheimer's beta-amyloid precursor protein in human cells: evidence for a role of the prohormone convertase PC7 in the constitutive alpha-secretase pathway.

The physiological maturation of the beta-amyloid precursor protein (betaAPP) leads to the secretion of a fragment termed APPalpha, after cleavage by a proteolytic enzyme called-secretase. In Alzheimer's disease, betaAPP undergoes exacerbated proteolytic attacks by beta- and gamma-secretases, which liberate a readily aggregatable 40-42-amino acid peptide called AP. We show here that overexpression of the prohormone convertase PC7 triggers increased secretion of APPalpha and lowers both Abeta40 and Abeta42 recoveries. Overexpression of alpha1-antitrypsin Portland (alpha1-PDX), which blocks mammalian precursor convertases of the constitutive secretory pathway, reverses the PC7-induced APPalpha increase as well as the decrease of Abeta40/42 in HEK293 cells. It is interesting that alpha1-PDX also lowers the level of APPalpha endogenously produced by mock-transfected HEK293 cells. Finally, a Jurkat clone stably expressing alpha1-PDX produces noticeably lower amounts of APPalpha. Therefore, this serpin affects endogenous a-secretase activity/pathway in distinct cell types. By contrast, alpha1-PDX does not alter the processing of presenilin 1 or its mutated congeners linked to some familial forms of Alzheimer's disease. Altogether, we demonstrate that a prohormone convertase participates in the alpha-secretase pathway of betaAPP maturation in human cells and concomitantly contributes to slowing the pathogenic route leading to the formation of Abeta. Our data strongly suggest that PC7 could fulfill such a role.     

Alkylation and cleavage of a DNA fragment with trioligonucleotide reagent

A possibility of site-directed chemical modification of a ssDNA fragment with "trioligonucleotide reagent" (TOR), consisting of a central oligonucleotide derivative carrying N-(2-chloroethyl)-N-(p-formylphenyl)-N-propyl-N-3-ydeneamino groups at both 5'- and 3'-thiophosphate ends and two border derivatives with 4-carbohydrazidephenyl groups at their 3'- and 5'-phosphate ends, respectively, is shown. Products of site-directed fragment cleavage, more abundant than the alkylation products, were found at 50 degrees C. The overall level of DNA modification by TOR reached 30% at a small excess of the oligonucleotide derivatives.     

Cathepsin cleavage potentiates the Ebola virus glycoprotein to undergo a subsequent fusion-relevant conformational change

Cellular entry of Ebola virus (EBOV), a deadly hemorrhagic fever virus, is mediated by the viral glycoprotein (GP). The receptor-binding subunit of GP must be cleaved (by endosomal cathepsins) in order for entry and infection to proceed. Cleavage appears to proceed through 50-kDa and 20-kDa intermediates, ultimately generating a key 19-kDa core. How 19-kDa GP is subsequently triggered to bind membranes and induce fusion remains a mystery. Here we show that 50-kDa GP cannot be triggered to bind to liposomes in response to elevated temperature but that 20-kDa and 19-kDa GP can. Importantly, 19-kDa GP can be triggered at temperatures ～10°C lower than 20-kDa GP, suggesting that it is the most fusion ready form. Triggering by heat (or urea) occurs only at pH 5, not pH 7.5, and involves the fusion loop, as a fusion loop mutant is defective in liposome binding. We further show that mild reduction (preferentially at low pH) triggers 19-kDa GP to bind to liposomes, with the wild-type protein being triggered to a greater extent than the fusion loop mutant. Moreover, mild reduction inactivates pseudovirion infection, suggesting that reduction can also trigger 19-kDa GP on virus particles. Our results support the hypothesis that priming of EBOV GP, specifically to the 19-kDa core, potentiates GP to undergo subsequent fusion-relevant conformational changes. Our findings also indicate that low pH and an additional endosomal factor (possibly reduction or possibly a process mimicked by reduction) act as fusion triggers.     

Proprotein conversion is determined by a multiplicity of factors including convertase processing, substrate specificity, and intracellular environment. Cell type-specific processing of human prorenin by the convertase PC1.

Proprotein and prohormone processing at pairs of basic residues is generally thought to be both tissue- and precursor-specific and to be developmentally regulated. Furin, PC1 (also called PC3), and PC2 represent three recently discovered subtilisin-like proteinases which cleave a number of precursors at the same pairs of basic residues normally processed in vivo. Using human prorenin as a model, we show that PC1 can process it to active renin in cells containing secretory granules, such as the somatomammotroph cell line GH4, but not in cells which lack granules, such as the Chinese hamster ovary or African green monkey kidney epithelial (BSC-40) cell lines. In contrast, in both cell types, human prorenin is not activated by either PC2 or furin. Using the vaccinia virus expression system, biosynthetic labeling experiments demonstrated that PC1 and PC2 are themselves cleaved intracellularly at pairs of basic residues and that these two proenzymes are processed to different extents independent of whether the cell line contains dense core secretory granules. Furthermore, we also show that the cells mostly secrete the cleaved forms of PC1 and PC2, and that intracellularly the pro- form of PC2 predominates. Our data demonstrate that propeptide removal from these enzymes, possibly leading to their activation, is not the only criterion which governs precursor processing.     

Insulin proteinase liberates from glucagon a fragment known to have enhanced activity against Ca2+ + Mg2+-dependent ATPase.

We find, contrary to previous reports, that substantial cleavage of glucagon by insulin proteinase occurs at only one region, namely the double-basic sequence -Arg17-Arg18-. Cleavage takes place almost exclusively between these two residues, liberating fragments glucagon-(1-17) and glucagon-(18-29). Others have shown that the fragment glucagon-(19-29) is 1000-fold more efficient compared with intact glucagon, at inhibiting the Ca2+-activated and Mg2+-dependent ATPase activity and the Ca2+ pump of liver plasma membranes. We show that this fragment is not liberated in detectable quantities by our insulin proteinase preparation. On the other hand, others have shown that glucagon-(18-29), though less active than glucagon-(19-29), was still 100-fold more active than glucagon itself in the above-mentioned system. Our observations represent the first demonstration of the release by insulin proteinase of a hormone fragment having enhanced activity, although it has yet to be shown that the activity of this fragment is important in vivo. Since the formation of glucagon-(19-29) from glucagon-(18-29) would involve merely removal of Arg18, a second enzyme might exist to provide the more active fragment.     

Evidence that platelet glycoprotein IIIa has a large disulfide-bonded loop that is susceptible to proteolytic cleavage

The proteolytic digestion of GPIIIa on intact platelets by chymotrypsin, thrombin, plasmin, trypsin, and staphylococcal V8 protease was monitored in immunoblot studies employing three different antibodies to GPIIIa, one of which was made against a 13-residue synthetic peptide containing the amino terminus of GPIIIa. Chymotrypsin, plasmin, and trypsin gave similar patterns, from which it could be inferred that the major products after extensive digestion were two-chain molecules composed of amino-terminal fragments of Mr approximately 17,000-18,000 disulfide bonded to carboxyl-terminal remnants of Mr approximately 58,000-70,000. These patterns suggest that GPIIIa contains a large disulfide-bonded loop of at least 325 amino acids that is susceptible to proteolytic cleavage, and that the 4 cysteine residues between residues 177 and 273 bond with each other. Such a structure can also account for the presence of the PIA1 epitope, which has recently been localized to a polymorphism at position 33 on these late digestion products. Thrombin did not proteolyze GPIIIa even at 2.5 units/ml. Still to be resolved is whether the minor immunoreactive GPIIIa bands found on normal platelets are related to in vivo or in vitro proteolysis and whether GPIIIa proteolysis plays a role in chymotrypsin-induced exposure of the GPIIb/IIIa receptor.     

Membrane proximal cleavage of L-selectin: identification of the cleavage site and a 6-kD transmembrane peptide fragment of L-selectin

Rapid downregulation of L-selectin expression occurs in response to leukocyte activation, and it has been speculated to be an integral process in the adhesion cascade leading to neutrophil recruitment to sites of inflammation. It has previously been proposed that L-selectin is proteolytically cleaved from the cell surface; however, the nature of the cleavage site has been unknown. We have produced polyclonal antisera against the extracellular domain and against the cytoplasmic domain of L-selectin. Both antisera immunoprecipitate the intact form of L-selectin from metabolically labeled phytohemagglutinin-stimulated lymphoblasts and peripheral blood neutrophils. In addition, the anti- cytoplasmic domain serum, but not the antiectodomain serum, immunoprecipitate a 6-kD species from PMA activated lymphoblasts and formylmethionylleucylphenylalanine-activated neutrophils. Conversely, the antiectodomain serum but not the anti-cytoplasmic domain serum immunoprecipitate a 68-kD soluble form of L-selectin from the supernatant of PMA-activated lymphoblasts. The appearance of the 6-kD species on activated cells correlated with the disappearance of the intact form of L-selectin and the appearance of the soluble form of L- selectin. A third polyclonal serum generated against the membrane proximal region of the ectodomain also reacted with the 6-kD species, indicating that this is a transmembrane peptide of L-selectin. That the 6-kD species is derived from L-selectin was confirmed by immunoprecipitation of the 6-kD species from L-selectin transfectants but not from mock transfectants. Radiochemical sequence analysis defined a cleavage site between Lys321 and Ser322, which would predict a transmembrane fragment consistent in size with the observed 6-kD fragment. A Ser-Phe-Ser motif adjacent to the cleavage site is conserved between human, mouse, and rat L-selectin, and a related motif is found proximal to transmembrane domains of other downregulated proteins, such as ACE, CD16-II, and TNF-RII, suggesting the possibility of a common recognition motif.     

Binding of C3 molecules to membranes via the SH-residue generated by the cleavage of a thioester bond can initiate complement activation

Dithiopyridine (DTP)-dipalmitoylphosphatidylethanolamine (DTP-DPPE) was incorporated into liposome membranes to prepare DTP-liposomes. The DTP-liposomes could be lysed by reaction with the alternative complement pathway of any kind of serum tested. Activation of the alternative complement pathway has been shown to be mediated by the binding of C3 molecules to DTP on the liposomes via the SH-residue generated by the cleavage of thioester bond in the alpha-chain of the molecules.     

A monoclonal antibody to a synthetic fragment of rabies virus glycoprotein binds ligands of the nicotinic cholinergic receptor

Rabies virus glycoprotein and snake venom curaremimetic neurotoxins share a region of high homology (30-45 for neurotoxins and 190-203 for the glycoprotein) in the regions that are believed to be responsible for binding the nicotinic acetylcholine receptor. Monoclonal antibodies raised to the 190-203 synthetic fragment of rabies virus glycoprotein were immobilized on a high performance affinity chromatography column and were able to bind neurotoxins. Toxins were displaced from the affinity column by elution at acidic pH and by affinity competition with acetylcholine at neutral pH. Furthermore, the affinity column proved to be useful for the purification of cholinergic ligands. Overall, these results indicate that the paratope of our monoclonal antibodies could behave as an 'internal image' of the nicotinic cholinergic receptor acetylcholine binding site.     

1H- and natural abundance 15N-NMR studies of a derivative of a rabies glycoprotein fragment

Using a combination of one- and two-dimensional methods, 1H- and 15N-nmr spectroscopy has been employed to perform the complete assignment and the structural determination of the immunogenic undecapeptide CTTTNSRGTTT in DMSO solution. Nuclear Overhauser enhancement spectroscopy experiments indicated the presence of secondary structures, mainly turn-like structures, which only represent a family, albeit a dominant one, of an ensemble of conformations available to the peptide. Since reverse turns may play an important role as intermediates in protein folding, the experimental observations described here may link the immunological and theoretical approaches to protein folding.     

Proteolytic cleavage of a self-antigen following xenobiotic-induced cell death produces a fragment with novel immunogenic properties

The heavy metal mercury elicits a genetically restricted autoantibody response in mice that targets the nucleolar autoantigen fibrillarin. HgCl2-induced cell death of macrophages resulted in the proteolytic cleavage of fibrillarin. A prominent feature of mercury-induced cell death was the generation of a 19-kDa fragment of fibrillarin that was not found following apoptotic or nonapoptotic cell death induced by stimuli other than mercury. Proteolysis of fibrillarin lacking cysteines, and therefore unable to bind mercury, also produced the 19-kDa fragment, suggesting that a mercury-fibrillarin interaction was not necessary for the unique cleavage pattern of this self-Ag. In contrast to immunization with full-length fibrillarin, the 19-kDa fragment produced anti-fibrillarin Abs with some of the properties of the HgCl2-induced anti-fibrillarin response. We propose that cell death following exposure to an autoimmunity-inducing xenobiotic can lead to the generation of novel protein fragments that may serve as sources of antigenic determinants for self-reactive T lymphocytes.     

Cell type-specific post-Golgi apparatus localization of a "resident" endoplasmic reticulum glycoprotein, glucosidase II

Glucosidase II, an asparagine-linked oligosaccharide processing enzyme, is a resident glycoprotein of the endoplasmic reticulum. In kidney tubular cells, in contrast to previous findings on hepatocytes, we found by light and electron microscopy immunoreactivity for glucosidase II predominantly in post-Golgi apparatus structures. The majority of immunolabel was in endocytotic structures beneath the plasma membrane. Immunoprecipitation confirmed presence of the glucosidase II subunit in purified brush border preparations. Kidney glucosidase II contained species carrying endo H-sensitive, high mannose as well as endo H-resistant oligosaccharide chains. Some species of glucosidase II contained sialic acid. The sialylated species were enzymatically active. This study demonstrates than an enzyme presumed to be a resident of the endoplasmic reticulum may show alternative localizations in some cell types.     

Inhibitory activity and structural characterization of a C-terminal peptide fragment derived from the prosegment of the proprotein convertase PC7

Mammalian proprotein convertases (PCs) belong to the family of recently discovered serine proteases responsible for the processing of a large number of precursor proteins into their active forms. The enzymatic activities of the convertases have been implicated in a variety of disease states, such as cancer and infectious and inflammatory diseases. Like many other proteases, PCs are also synthesized as inactive proenzymes with N-terminal extensions as their prosegments. Here, we present the inhibitory activities of a number of "putative" interfacial peptide fragments derived from the proregion of PC7. We found that a peptide fragment corresponding to the C-terminal region (residues 81p-104p, or C24: E(1)-A-V-L-A-K-H-E-A-V-R-W-H-S-E-Q-R-L-L-K-R-A-K-R(24)) of the PC7 prosegment displays a strong inhibition (K(i) = 7 nM) of the PC7 enzyme comparable to that of the full-length (104 residue) prosegment. The same 24 residue peptide shows significantly populated helical conformations in an aqueous solution close to the physiological condition. Structure calculations driven by NOE distance restraints revealed a slightly kinked helical conformation for the entire peptide, characterized by many side-chain/side-chain interactions including those involving charged residues E8-R11-E15 and hydrophobic residues W12 and L19. These results suggest that the C-terminal region of the prosegment of PC7 may play a dominant role in conferring the inhibitory potency to the cognate enzyme and this strong inhibitory activity may be a direct consequence of the folded conformation of the peptide fragment in solution. We surmise that such a structure-function correlation for an inhibitory peptide could lead to the design and discovery of molecules mimicking the specific interactions of the PC prosegments for their cognate proteases.     

Structure of a hyper-cleavable monomeric fragment of phage lambda repressor containing the cleavage site region

The key event in the switch from lysogenic to lytic growth of phage lambda is the self-cleavage of lambda repressor, which is induced by the formation of a RecA-ssDNA-ATP filament at a site of DNA damage. Lambda repressor cleaves itself at the peptide bond between Ala111 and Gly112, but only when bound as a monomer to the RecA-ssDNA-ATP filament. Here we have designed a hyper-cleavable fragment of lambda repressor containing the hinge and C-terminal domain (residues 101-229), in which the monomer-monomer interface is disrupted by two point mutations and a deletion of seven residues at the C terminus. This fragment crystallizes as a monomer and its structure has been determined to 1.8 A resolution. The hinge region, which bears the cleavage site, is folded over the active site of the C-terminal oligomerization domain (CTD) but with the cleavage site flipped out and exposed to solvent. Thus, the structure represents a non-cleavable conformation of the repressor, but one that is poised for cleavage after modest rearrangements that are presumably stabilized by binding to RecA. The structure provides a unique snapshot of lambda repressor in a conformation that sheds light on how its self-cleavage is tempered in the absence of RecA, as well as a framework for interpreting previous genetic and biochemical data concerning the RecA-mediated cleavage reaction.