A Slow Maturation of a Cysteine Protease with a Granulin Domain in the Vacuoles of Senescing Arabidopsis Leaves

Arabidopsis RD21 is a cysteine protease of the papain family. Unlike other members of the papain family, RD21 has a C-terminal extension sequence composed of two domains, a 2-kD proline-rich domain and a 10-kD domain homologous to animal epithelin/granulin family proteins. The RD21 protein was accumulated as 38- and 33-kD proteins in Arabidopsis leaves. An immunoblot showed that the 38-kD protein had the granulin domain, whereas the 33-kD protein did not. A pulse-chase experiment with Bright-Yellow 2 transformant cells expressing RD21 showed that RD21 was synthesized as a 57-kD precursor and was then slowly processed to make the 33-kD mature protein via the 38-kD intermediate. After a 12-h chase, the 38-kD intermediate was still detected in the cells. These results indicate that the N-terminal propeptide was first removed from the 57-kD precursor, and the C-terminal granulin domain was then slowly removed to yield the 33-kD mature protein. Subcellular fractionation of the Bright-Yellow 2 transformant showed that the intermediate and mature forms of RD21 were localized in the vacuoles. Under the acidic conditions of the vacuolar interior, the intermediate was found to be easily aggregated. The intermediate and the mature protein were accumulated in association with leaf senescence. Taken together, these results indicate that the intermediate of RD21 was accumulated in the vacuoles as an aggregate, and then slowly matured to make a soluble protease by removing the granulin domain during leaf senescence.     

A Kunitz-type cysteine protease inhibitor from cauliflower and Arabidopsis

A Kunitz-type protease inhibitor co-purified from cauliflower florets with a granulin domain cysteine protease that cleaved barley proaleurain to yield a molecular form the same size as that for mature aleurain. The purified cauliflower protease required treatment with SDS detergent to become active. This observation raised the question of whether the protease inhibitor might have the ability to interact with the granulin domain protease. Here we express an Arabidopsis homolog of the protease inhibitor as a recombinant protein and demonstrate that it is a potent inhibitor of the recombinant proaleurain maturation protease and of papain when assayed at pH 4.5 but not at pH 6.3. In a pull-down assay, the inhibitor bound tightly to papain, but only weakly to the aspartate protease pepsin. When the cauliflower protease inhibitor was transiently expressed in tobacco suspension culture protoplasts, it colocalized with BP-80, a vacuolar sorting receptor that interacts with proaleurain and traffics to prevacuolar compartments for lytic vacuoles. Our results indicate that the cauliflower and Arabidopsis protease inhibitors would traffic through cellular compartments where proaleurain also traffics. Their ability to inhibit a cysteine protease implicated in maturation of proaleurain to active form at the acidic pH found in vacuoles raises the possibility that they could participate in regulating activation of aleurain.     

Post-translational regulation and trafficking of the granulin-containing protease RD21 of Arabidopsis thaliana

RD21-like proteases are ubiquitous, plant-specific papain-like proteases typified by carrying a C-terminal granulin domain. RD21-like proteases are involved in immunity and associated with senescence and various types of biotic and abiotic stresses. Here, we interrogated Arabidopsis RD21 regulation and trafficking by site-directed mutagenesis, agroinfiltration, western blotting, protease activity profiling and protein degradation. Using an introduced N-glycan sensor, deglycosylation experiments and glyco-engineered N. benthamiana plants, we show that RD21 passes through the Golgi where it becomes fucosylated. Our studies demonstrate that RD21 is regulated at three post-translational levels. Prodomain removal is not blocked in the catalytic Cys mutant, indicating that RD21 is activated by a proteolytic cascade. However, RD21 activation in Arabidopsis does not require vacuolar processing enzymes (VPEs) or aleurain-like protease AALP. In contrast, granulin domain removal requires the catalytic Cys and His residues and is therefore autocatalytic. Furthermore, SDS can (re-)activate latent RD21 in Arabidopsis leaf extracts, indicating the existence of a third layer of post-translational regulation, possibly mediated by endogenous inhibitors. RD21 causes a dominant protease activity in Arabidopsis leaf extracts, responsible for SDS-induced proteome degradation.     

Characterization of an alkaline protease associated with a granulosis virus of Plodia interpunctella.

An alkaline protease was found to be associated with the granulosis virus of the Indian meal moth. Plodia interpunctella. The protease was located within the protein matrix of the occluded virus and hydrolyzed the major constituent of this matrix, a 28,000-dalton protein (granulin), to a mixture of polypeptides ranging in molecular weight from 10,000 to 27,000. A rapid, sensitive assay for the protease was developed using radioactively labeled granulosis virus as substrate. With this assay, the proteolytic activity could be detected by measuring the release of acid-soluble peptides from the labeled virus. The protease had a pH optimum of 10.5 and a temperature optimum of 40 degrees C and was inhibited by diisopropyl phosphorofluoridate, phenylmethylsulfonyl fluoride, and L-(1-tosylamido-2-phenyl) ethyl chloromethyl ketone. Purification of the protease from matrix protein was achieved by anion-exchange and gel permeation chromatography. The molecular weight of the isolated protease, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, was approximately 14,000.     

A cysteine protease gene is expressed early in resistant potato interactions with Phytophthora infestans

A potato cysteine protease (cyp) cDNA expressed at an early stage of an incompatible interaction with Phytophthora infestans was isolated. Both the nucleotide and deduced amino acid sequences are highly homologous to those of a tomato cysteine protease, CYP1. Striking protein similarity to all known cathepsins in animals, particularly cathepsin K, was also observed. However, unlike cathepsins, a granulin binding domain is located near the carboxyl terminus of the putative CYP protein. In animals, granulins bind to receptors in the plasma membrane and signal cell growth and division. A ribonuclease protection assay demonstrated that the cyp gene is tightly regulated and is induced 15 h post inoculation with P. infestans in potato leaves either with high field resistance or in which a resistance (R) gene is activated. We conclude that a common signaling pathway is activated in each form of resistance.     

Epithelin/granulin growth factors: extracellular cofactors for HIV-1 and HIV-2 Tat proteins

Epithelin/granulin growth factor is synthesized as a 593 amino acid precursor protein that contains 7.5 imperfectly conserved repeats of approximately 57 amino acids. Processed epithelin/granulin peptides have been isolated from vertebrate/invertebrate species and are growth factors implicated in epithelial and haemic cell function. Here they are identified as Human Immunodeficiency Virus (HIV) Tat binding proteins using the yeast two-hybrid assay. Intracellularly in yeast, mutation of selected cysteines in an epithelin/granulin dimeric repeat caused loss of binding to Tat exon 1. In vitro binding of HIV-1 and HIV-2 Tat to epithelin/granulin dimeric and monomeric repeats was also observed by GST-glutathione bead "pulldown" assays. Because Tat is actively secreted from HIV-infected cells and has been shown to serve as a mitogenic factor for angiogenesis and for Kaposi-like cells, our observations suggest that epithelin/granulin growth factors may function as biologically important extracellular Tat co-factors.     

Structure and chromosomal location of the human granulin gene.

Granulins are a family of cysteine rich polypeptides some of which have growth modulatory activity. We showed previously that the granulins are encoded within the same precursor consisting of seven granulin domains arranged in tandem. Here we report the chromosomal location and structural organization of the protein coding region of the granulin gene. The granulin gene was assigned to chromosome 17 using DNA from human-hamster somatic cell hybrids. The protein-coding region of the granulin gene was shown to comprise 12 exons covering about 3700 bp. Each tandem granulin repeat is encoded by two non-equivalent exons, a configuration unique to the granulins that would permit the formation of hybrid granulin-like proteins by alternate splicing.     

Subclassification and biochemical analysis of plant papain-like cysteine proteases displays subfamily-specific characteristics

Papain-like cysteine proteases (PLCPs) are a large class of proteolytic enzymes associated with development, immunity, and senescence. Although many properties have been described for individual proteases, the distribution of these characteristics has not been studied collectively. Here, we analyzed 723 plant PLCPs and classify them into nine subfamilies that are present throughout the plant kingdom. Analysis of these subfamilies revealed previously unreported distinct subfamily-specific functional and structural characteristics. For example, the NPIR and KDEL localization signals are distinctive for subfamilies, and the carboxyl-terminal granulin domain occurs in two PLCP subfamilies, in which some individual members probably evolved by deletion of the granulin domains. We also discovered a conserved double cysteine in the catalytic site of SAG12-like proteases and two subfamily-specific disulfides in RD19A-like proteases. Protease activity profiling of representatives of the PLCP subfamilies using novel fluorescent probes revealed striking polymorphic labeling profiles and remarkably distinct pH dependency. Competition assays with peptide-epoxide scanning libraries revealed common and unique inhibitory fingerprints. Finally, we expand the detection of PLCPs by identifying common and organ-specific protease activities and identify previously undetected proteases upon labeling with cell-penetrating probes in vivo. This study provides the plant protease research community with tools for further functional annotation of plant PLCPs.     

Purification of granulin-like polypeptide from the blood-sucking leech, Hirudo nipponia.

A cysteine-rich (approximately 20%), low molecular weight (MW 6 kDa) polypeptide has been isolated from the Korean blood-sucking leech, Hirudo nipponia. From its amino acid composition and N-terminal amino acid sequence analysis, the new protein is similar to granulin (or epithelin), and so it has been named leech granulin. The leech granulin behaved as a thrombin inhibitor in the purification steps of size-exclusion, ion-exchange, chromatofocusing, and reverse-phase high-performance liquid chromatography. The leech granulin is an acidic peptide (pI 3.75) containing high cysteine residues with a unique sequence similar to granulins or epithelins isolated from other organisms. For the first time, a granulin-like polypeptide having thrombin inhibitory activity has been isolated from a leech species.     

Granulins, a novel class of peptide from leukocytes

We report the isolation and characterization of a novel class of leukocyte peptides with possible cytokine-like activities which we call granulins. They are cystine-rich with molecular weights of approximately 6 Kda, except for granulin D, which appears to be a dimer. We present the sequence of one member of this family, a 56 residue peptide, granulin A, and amino-terminal sequences for three other granulins from human peripheral leukocytes. A fifth related peptide was isolated and partially sequenced from rat bone marrow, suggesting that at least some of the granulin in peripheral leukocytes is preformed in the marrow. Rat granulin, and human granulin A, are closely related, showing that the granulin structures are highly conserved between species.     

The Evolution of the Secreted Regulatory Protein Progranulin

Progranulin is a secreted growth factor that is active in tumorigenesis, wound repair, and inflammation. Haploinsufficiency of the human progranulin gene, GRN, causes frontotemporal dementia. Progranulins are composed of chains of cysteine-rich granulin modules. Modules may be released from progranulin by proteolysis as 6kDa granulin polypeptides. Both intact progranulin and some of the granulin polypeptides are biologically active. The granulin module occurs in certain plant proteases and progranulins are present in early diverging metazoan clades such as the sponges, indicating their ancient evolutionary origin. There is only one Grn gene in mammalian genomes. More gene-rich Grn families occur in teleost fish with between 3 and 6 members per species including short-form Grns that have no tetrapod counterparts. Our goals are to elucidate progranulin and granulin module evolution by investigating (i): the origins of metazoan progranulins (ii): the evolutionary relationships between the single Grn of tetrapods and the multiple Grn genes of fish (iii): the evolution of granulin module architectures of vertebrate progranulins (iv): the conservation of mammalian granulin polypeptide sequences and how the conserved granulin amino acid sequences map to the known three dimensional structures of granulin modules. We report that progranulin-like proteins are present in unicellular eukaryotes that are closely related to metazoa suggesting that progranulin is among the earliest extracellular regulatory proteins still employed by multicellular animals. From the genomes of the elephant shark and coelacanth we identified contemporary representatives of a precursor for short-from Grn genes of ray-finned fish that is lost in tetrapods. In vertebrate Grns pathways of exon duplication resulted in a conserved module architecture at the amino-terminus that is frequently accompanied by an unusual pattern of tandem nearly identical module repeats near the carboxyl-terminus. Polypeptide sequence conservation of mammalian granulin modules identified potential structure-activity relationships that may be informative in designing progranulin based therapeutics.     

Activity‐based proteomics reveals nine target proteases for the recombinant protein‐stabilizing inhibitor SlCYS8 in Nicotiana benthamiana

Co‐expression of protease inhibitors like the tomato cystatin SlCYS8 is useful to increase recombinant protein production in plants, but key proteases involved in protein proteolysis are still unknown. Here, we performed activity‐based protein profiling to identify proteases that are inhibited by SlCYS8 in agroinfiltrated Nicotiana benthamiana. We discovered that SlCYS8 selectively suppresses papain‐like cysteine protease (PLCP) activity in both apoplastic fluids and total leaf extracts, while not affecting vacuolar‐processing enzyme and serine hydrolase activity. A robust concentration‐dependent inhibition of PLCPs occurred in vitro when purified SlCYS8 was added to leaf extracts, indicating direct cystatin–PLCP interactions. Activity‐based proteomics revealed that nine different Cathepsin‐L/‐F‐like PLCPs are strongly inhibited by SlCYS8 in leaves. By contrast, the activity of five other Cathepsin‐B/‐H‐like PLCPs, as well as 87 Ser hydrolases, was unaffected by SlCYS8. SlCYS8 expression prevented protein degradation by inhibiting intermediate and mature isoforms of granulin‐containing proteases from the Resistant‐to‐Desiccation‐21 (RD21) PLCP subfamily. Our data underline the key role of endogenous PLCPs on recombinant protein degradation and reveal candidate proteases for depletion strategies.     

Effects of Substituting Granulin or a Granulin-Polyhedrin Chimera for Polyhedrin on Virion Occlusion and Polyhedral Morphology in Autographa californica Multinucleocapsid Nuclear Polyhedrosis Virus

Substitution of granulin from the Trichoplusia ni granulosis virus (TnGV) for polyhedrin of the Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) yielded a few very large (2 to 5 μm) cuboidal inclusions in the cytoplasm and nucleus of infected cells. These polyhedra lacked the beveled edges characteristic of wild-type AcMNPV polyhedra, contained fractures, and occluded few virions. Placing a nuclear localization signal (KRKK) in granulin directed more granulin to the nucleus and resulted in more structurally uniform cuboidal inclusions in which no virions were observed. A granulin-polyhedrin chimera produced tetrahedral occlusions with more virions than granulin inclusions but many fewer than wild-type polyhedra. Despite the unusual structure of the granulin and granulin-polyhedrin inclusions, they interacted with AcMNPV p10 fibrillar structures and electron-dense spacers that are precursors of the polyhedral calyx. The change in inclusion shape obtained with the granulin-polyhedrin chimera demonstrates that the primary amino acid sequence affects occlusion body shape, but the large cuboidal inclusions formed by granulin indicate that the amino acid sequence is not the only determinant. The failure of granulin or the granulin-polyhedrin chimera to properly occlude AcMNPV virions suggests that specific interactions occur between polyhedrin and other viral proteins which facilitate normal virion occlusion and occlusion body assembly and shape in baculoviruses.     

Stimulation of PC cell-derived growth factor (epithelin/granulin precursor) expression by estradiol in human breast cancer cells

PC cell-derived growth factor (PCDGF) is an 88 kDa glycosylated protein isolated from a highly tumorigenic mouse teratoma derived cell line which is similar to the epithelin/granulin precursor. Using Northern blot and western blot analyses, we detect the expression of PCDGF mRNA and protein in MCF-7 human breast cancer cells. We show that 17-beta-estradiol stimulates PCDGF mRNA and protein expression in a time and dose-dependent manner. The stimulation of PCDGF expression by 17-beta-estradiol was observed as early as 4 hours and reached a maximum at 12 hours. Maximal stimulation of PCDGF mRNA and protein expression by 17-beta-estradiol was observed at a concentration of 10(-8) M. The stimulation of PCDGF expression by 17-beta-estradiol was completely inhibited by treatment with actinomycin D and with the antiestrogen 4-hydroxytamoxifen. The stimulation of PCDGF expression was also demonstrated in another human estrogen-responsive cell line T47D. The results presented here provide evidence of a novel estradiol responsive gene product in human breast cancer cell lines and give information about the hormonal control of epithelin/granulin (PCDGF) expression in these cells.     

Development and application of mass spectrometric methods for the analysis of progranulin N-glycosylation

PGRN is a modular protein with 7 1/2 repeats of the granulin domain separated by short spacer sequences. Elevated expression of PGRN is associated with cancer growth, while mutations of PGRN cause frontotemporal lobar degeneration (FTLD), an early onset form of dementia. PGRN is a glycoprotein, containing five N-glycosylation consensus sequons, three of which fall within granulin domains. A method tailored to enable detailed analysis of the PGRN oligosaccharides and glycopeptides has been developed. The approach involves in-gel deglycosylation using peptide-N-glycosidase F (PNGase F) followed by permethylation of the released oligosaccharides. Permethylation was applied for rapid sample clean-up and to improve sensitivity of MS detection and mass spectrometric fragmentation. Reversed-phase monolithic LC–ESI–MS/MS was used for analysis of permethylated oligosaccharides, enabling structural characterization of released N-linked glycans in one chromatographic run. In-gel tryptic digestion was further applied to the gel pieces containing deglycosylated protein, for N-glycosylation site determination. In addition, glycopeptides were produced using in-solution pronase digestion to identify species of N-glycan attached at particular sites. The method developed was applied to progranulin (PGRN) to characterize the structures of the released glycans and to identify the sites of glycosylation. Glycosylation of four out of five potential PGRN N-glycosylation consensus sites was demonstrated (the final one remains undetermined), with one of the four observed to be partially occupied. Two of the observed glycosylation sites occur within granulin domains, which may have important implications for understanding the structural basis of PGRN action.     

An effector-targeted protease contributes to defense against Phytophthora infestans and is under diversifying selection in natural hosts

Since the leaf apoplast is a primary habitat for many plant pathogens, apoplastic proteins are potent, ancient targets for apoplastic effectors secreted by plant pathogens. So far, however, only a few apoplastic effector targets have been identified and characterized. Here, we discovered that the papain-like cysteine protease C14 is a new common target of EPIC1 and EPIC2B, two apoplastic, cystatin-like proteins secreted by the potato (Solanum tuberosum) late blight pathogen Phytophthora infestans. C14 is a secreted protease of tomato (Solanum lycopersicum) and potato typified by a carboxyl-terminal granulin domain. The EPIC-C14 interaction occurs at a wide pH range and is stronger than the previously described interactions of EPICs with tomato defense proteases PIP1 and RCR3. The selectivity of the EPICs is also different when compared with the AVR2 effector of the fungal tomato pathogen Cladosporium fulvum, which targets PIP1 and RCR3, and only at apoplastic pH. Importantly, silencing of C14 increased susceptibility to P. infestans, demonstrating that this protease plays a role in pathogen defense. Although C14 is under conservative selection in tomato, it is under diversifying selection in wild potato species (Solanum demissum, Solanum verrucosum, and Solanum stoliniferum) that are the natural hosts of P. infestans. These data reveal a novel effector target in the apoplast that contributes to immunity and is under diversifying selection, but only in the natural host of the pathogen.