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At least 19 recordsLinked to original sources

PubMed1998

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 micron) 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.

Amino Acid Sequence↗

PubMed2005

Granulin and granulin repeats interact with the Tat.P-TEFb complex and inhibit Tat transactivation.

The cellular positive transcription elongation factor b (P-TEFb), containing cyclin T1 and cyclin-dependent kinase 9 (CDK9), interacts with the human immunodeficiency virus, type 1 (HIV-1) regulatory protein Tat to enable viral transcription and replication. Cyclin T1 is an unusually long cyclin and is engaged by cellular regulatory proteins. Previous studies showed that the granulin/epithelin precursor (GEP) binds the histidine-rich region of cyclin T1 and inhibits P-TEFb function. GEP is composed of repeats that vary in sequence and properties. GEP also binds directly to Tat. Here we show that GEP and some of its constituent granulin repeats can inhibit HIV-1 transcription via Tat without directly binding to cyclin T1. The interactions of granulins with Tat and cyclin T1 differ with respect to their binding sites and divalent cation requirements, and we identified granulin repeats that bind differentially to Tat and cyclin T1. Granulins DE and E bind Tat but do not interact directly with cyclin T1. These granulins are present in complexes with Tat and P-TEFb in which Tat forms a bridge between the cellular proteins. Granulins DE and E repress transcription from the HIV-1 LTR and gene expression from the viral genome, raising the possibility of developing granulin-based inhibitors of viral infection.

Amino Acid Sequence↗

PubMed1992

Isolation and sequence of the granulin precursor cDNA from human bone marrow reveals tandem cysteine-rich granulin domains.

Granulins are candidate growth factors recently discovered in human and rat inflammatory leukocytes and bone marrow. Two granulin homologs, epithelin 1 and 2, occur in the rat kidney. Epithelin 1, which is probably identical to rat leukocyte granulin, exhibits proliferative and antiproliferative effects on epithelial cells in vitro. Here we show by cDNA analysis that the prepropeptide for the human granulins is a 593-residue glycoprotein, containing seven tandem repeats of the 12-cysteine granulin domain. By Northern blot analysis, gene expression was seen in myelogenous leukemic cell lines of promonocytic, promyelocytic, and proerythroid lineage, in fibroblasts and was seen very strongly in epithelial cell lines. Some epithelial cell lines respond to the mature peptide and express the gene. Among tissues examined, the kidney had the highest levels of granulin mRNA.

Amino Acid Sequence↗

PubMed1993

Isolation and primary structure of the three major forms of granulin-like peptides from hematopoietic tissues of a teleost fish (Cyprinus carpio).

Granulins are cysteine-rich polypeptides purified from human and rat hematopoietic cells and structurally related to the epithelin family of growth modulatory factors. A prototypic form of granulin was isolated from the hematopoietic organs of teleost fish (Belcourt, D., and Bennett, H.P.J. (1988) J. Cell Biol. 107, 629 (abstr.)). This study reports the structure of three granulins purified from the spleen and head kidney of the carp (Cyprinus carpio). Ion-spray mass spectrometric analysis of granulin-1, -2, and -3 corroborated the observed primary structures and demonstrated that each 57-residue-peptide was monomeric in nature with all cysteines linked via intramolecular disulfide bridges. A comparison of the carp granulin sequences demonstrates that granulins 2 and 3 are most closely related with sequence variations occurring primarily toward the amino terminus. A rabbit polyclonal antibody was raised against carp granulin-1 to develop a radioimmunoassay for this peptide, which showed no significant cross-reactivity with granulin-2 and -3. The distribution of carp granulin-1 was studied by screening purified tissue extracts for immunoreactivity using reversed-phase high performance liquid chromatography. A single form of immunoreactive granulin-1 was identified in all carp tissues studied including spleen, head kidney, heart, skin, gills, and gut. These studies have established that members of the granulin/epithelin family are found in a lower vertebrate and may serve important growth modulatory functions throughout the vertebrate kingdom.

Amino Acid Sequence↗

PubMed2006

A novel hematopoietic granulin induces proliferation of goldfish (Carassius auratus L.) macrophages.

Granulins are a group of highly conserved growth factors that have been described from a variety of organisms spanning the metazoa. In this study, goldfish granulin was one of the most commonly identified transcripts in the differential cross-screening of macrophage cDNA libraries and was preferentially expressed in proliferating macrophages. Unlike mammalian granulins, which possess 7.5 repeats of a characteristic signature of 12 cysteine residues, the goldfish granulin encoded a putative peptide possessing only 1.5 cysteine repeats. Northern blot and real-time PCR analyses indicated that goldfish granulin was expressed only in the hematopoietic tissues of the goldfish, specifically the kidney and spleen, and in activated peripheral blood mononuclear cells. We expressed granulin using a prokaryotic expression system and produced an affinity-purified rabbit anti-goldfish granulin IgG. Recombinant goldfish granulin induced a dose-dependent proliferative response of goldfish macrophages that was inversely related to the myeloid differentiation stage of the cells studied. The highest proliferative response was observed in macrophage progenitor cells and monocytes. This proliferative response of macrophages was abrogated by the addition of anti-granulin IgG. These results indicate that goldfish granulin is a growth factor that positively modulates cell proliferation at distinct junctures of macrophage differentiation.

Amino Acid Sequence↗

PubMed1992

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.

Alternative Splicing↗

PubMed1995

Immunocytochemical localization of granulin-1 to mononuclear phagocytic cells of the teleost fish Cyprinus carpio and Carassius auratus.

A new class of low-molecular-weight cysteine-rich regulatory growth factors, designated granulins, has been isolated from hematopoietic tissues of a teleost fish (Cyprinus carpio) and structurally characterized. Granulin-1, the predominant form found in carp spleen, was used to raise polyclonal antibodies in rabbits and to establish a radioimmunoassay. This permitted preliminary tissue distribution studies of granulin-1 to be undertaken in carp (Cyprinus carpio) and goldfish (Carassius auratus). Granulin-1 immunoreactivity was found in the melanomacrophage centers of the spleen and head kidney. Carp tissues anatomically involved in the first line of defense against infection, including skin, gills, gut, and also heart, showed intense granulin-1 immunoreactive staining within presumptive macrophage cells. Granulin-1 immunoreactive macrophages prepared from goldfish spleen and head kidney adhered to glass slides, actively phagocytosed carbon particles, and contained granulin-1 immunoreactivity as well as abundant endogenous peroxidase activity. This study demonstrates that granulin-1 is synthesized and stored in macrophages/monocytes of spleen, head kidney, and peripheral tissues of teleost fish.

Acid Phosphatase↗

PubMed1990

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.

Amino Acid Sequence↗

PubMed2003

The growth factor granulin interacts with cyclin T1 and modulates P-TEFb-dependent transcription.

Cyclin T1, together with the kinase CDK9, is a component of the transcription elongation factor P-TEFb which binds the human immunodeficiency virus type 1 (HIV-1) transactivator Tat. P-TEFb facilitates transcription by phosphorylating the carboxy-terminal domain (CTD) of RNA polymerase II. Cyclin T1 is an exceptionally large cyclin and is therefore a candidate for interactions with regulatory proteins. We identified granulin as a cyclin T1-interacting protein that represses expression from the HIV-1 promoter in transfected cells. The granulins, mitogenic growth factors containing repeats of a cysteine-rich motif, were reported previously to interact with Tat. We show that granulin formed stable complexes in vivo and in vitro with cyclin T1 and Tat. Granulin bound to the histidine-rich domain of cyclin T1, which was recently found to bind to the CTD, but not to cyclin T2. Binding of granulin to P-TEFb inhibited the phosphorylation of a CTD peptide. Granulin expression inhibited Tat transactivation, and tethering experiments showed that this effect was due, at least in part, to a direct action on cyclin T1 in the absence of Tat. In addition, granulin was a substrate for CDK9 but not for the other transcription-related kinases CDK7 and CDK8. Thus, granulin is a cellular protein that interacts with cyclin T1 to inhibit transcription.

3T3 Cells↗

PubMed1997

Isolation and N-terminal sequence of multiple forms of granulins in human urine.

Three molecular forms of granulins (also known as epithelins) were isolated, for the first time, in human urine. Their N-terminal sequences, which have also been determined, are identical to those of granulins A and B, previously isolated from human leukocytes, and of granulin F, never isolated before but whose primary structure is known on the basis of the cDNA sequence. The urinary molecules, which show a molecular weight of about 6.5 kDa, are most likely produced by a posttranslational proteolytic processing occurring at the level of the kidney, which appears to be the organ richest in granulin precursor mRNA. The molecular events underlying the precursor processing are unknown, even though the involvement of the protease kallikrein, an enzyme thought to be responsible for the processing of several polypeptidic growth factor precursors, could be hypothesized. Granulins, however, do not show antikallikrein activity. The presence in human urine of isoform F, previously not identified from other human sources, seems to support the hypothesis that mature forms of granulins are generated by an organ-specific precursor processing, on the basis of particular physiological requirements, and to suggest also that this isoform may play "in vivo" an important and specific role in the epithelial cells of the human kidney.

Amino Acid Sequence↗

PubMed1999

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.

Amino Acid Sequence↗

PubMed2000

Comparative analysis of the granulin regions of the Phthorimaea operculella and Spodoptera littoralis granuloviruses.

The nucleotide sequence of two cloned restriction fragments encompassing the granulin genes from the granuloviruses of the potato tuber moth, Phthorimaea operculella, PhopGV, and the Egyptian cotton leaf worm, Spodoptera littoralis, SpliGV, have been determined. Although both viruses are able to infect the same Ph. operculella cell line, their granulins do not cluster in the same phylogenetic branches. PhopGV ganulin is closely related to Cydia pomonella GV (CpGV) and Cryptophlebia leucotreta GV (ClGV) (95.2 and 94% identity at the aminoacid level), while SpliGV granulin falls close to Trichoplusia ni GV and Xestia c-nigrum GV (91.6 and 92.0% respectively). The gene organization around the granulins reflects this clustering. Upstream the PhopGV granulin, an ORF belonging to the ME53 gene family (as ORF 124R of CpGV and 909 of ClGV) is present, while no equivalent ORF is found in this region in SpliGV. Downstream the granulin, both viruses present a gene homologous to the Autographa californica nucleopolyhedrovirus (AcMNPV) ORF 9 followed by a Protein Kinase (AcMNPV ORF10). The structure of this region seems thus conserved not only among nucleopolyhedroviruses but also in at least some granuloviruses.

Animals↗

PubMed2001

A peptide derived from the C-terminal part of a plant cysteine protease folds into a stack of two beta-hairpins, a scaffold present in the emerging family of granulin-like growth factors.

A 35 amino acid residue peptide corresponding to the N-terminal subdomain of the granulin-like repeat from rice oryzain beta was synthesized and regioselectively oxidized to produce a species with a [1-3, 2-4] disulfide-pairing pattern. The resulting peptide was studied in solution using NMR and was shown to adopt the tertiary topology of a stack of two beta-hairpins found in the emerging family of granulin-like growth factors. Because of the longer second beta-hairpin, the overall conformation of the peptide is somewhat more flexible than that of its well-structured carp granulin-1 analog. Except for the cysteine alignment, there is very little sequence homology between granulin-like growth factors from the animal kingdom and the granulin-like repeats at the C-termini of plant cysteine proteases. Therefore, the stack of two beta-hairpins may be a conserved three-dimensional organization of the granulin-like repeats from evolutionary distant sources with a significant role in specific protein-protein interactions.

Amino Acid Sequence↗

PubMed2000

Identification of a human glioma-associated growth factor gene, granulin, using differential immuno-absorption.

Identification of the genes that are differentially expressed in brain tumor cells but not in normal brain cells is important for understanding the molecular basis of these neurological cancers and for defining possible targets for therapeutic intervention. In an effort to discover potentially antigenic proteins that may be involved in the malignant transformation and progression of human glioblastomas, a novel antibody-based approach was developed to identify and isolate gene products that are expressed in brain tumors versus normal brain tissue. Using this method, whereby tumor-specific antibodies were isolated and used to screen a glioblastoma cDNA expression library, 28 gene products were identified. Nine of these clones had homology to known gene products, and 19 were novel. The expression of these genes in multiple different human gliomas was then evaluated by cDNA microarray hybridization. One of the isolated clones had consistently higher levels of expression (3-30-fold) in brain tumors compared with normal brain. Northern blot analysis and in situ hybridization confirmed this differential overexpression. cDNA sequence analysis revealed that this gene was identical to a relatively new class of growth regulators known as granulins, which have tertiary structures resembling the epidermal growth factor-like proteins. The 2.1-kb granulin mRNA was expressed predominantly in glial tumors, with lower levels in spleen, kidney, and testes, whereas expression was not detected in non-tumor brain tissues. Functional assays using [3H]thymidine incorporation indicated that granulin may be a glial mitogen, as addition of synthetic granulin peptide to primary rat astrocytes and three different early-passage human glioblastoma cultures increased cell proliferation in vitro, whereas increasing concentrations of granulin antibody inhibited cell growth in a dose-dependent manner. The differential expression pattern, tissue distribution, and implication of this glioma-associated molecule in growth regulation suggest a potentially important role for granulin in the pathogenesis and/or malignant progression of primary brain neoplasms.

Animals↗

PubMed1997

Identification and sequence analyses of the granulin gene of Choristoneura fumiferana granulovirus.

The nucleotide sequence of the granulin gene of Choristoneura fumiferana granulovirus (CfGV) was determined. The gene encodes a protein of 248 amino acids with a predicted Mr of 29.299 kDa. The granulin genes of Trichoplusia ni, Pieris brassicae and Cryptophlebia leucotreta granuloviruses showed homologies ranging from 76.7-80.5% for nucleotide sequences and 84.2-88.3% for amino acid sequences when compared to CfGV. The secondary structure of CfGV granulin protein, including the hydrophilic (polar) and hydrophobic (basic) regions, was predicted and found to be similar to other granulins. A very late baculovirus promoter motif, ATAAG, was found within the putative promoter region of the CfGV granulin gene.

Amino Acid Sequence↗

PubMed2000

Molecular characterization and phylogenetic analysis of the Harrisina brillians granulovirus granulin gene.

Well over 100 isolates of granulosis viruses (GVs), genus Granulovirus (family Baculoviridae), have been reported, all from lepidopterous insects. Three types of GVs are recognized, those of Type 1, which attack the fat body, Type 2, which attack most tissues, and Type 3, which attack only the midgut epithelium. To determine whether a correlation exists between tissue tropism and lepidopteran family phylogeny, the granulin gene of the Harrisina brillians (HbGV), a virus that attacks the midgut epithelium of H. brillians (family Zygaenidae) was cloned, sequenced, characterized, and compared with granulin genes of GVs that attack species of Tortricidae, Pieridae, and Noctuidae. The HbGV granulin gene encoded a peptide of 248 amino acids with a predicted Mr of 29.6 kDa, and shared a significant level of homology with other granulin (81-95% identical and 90-98% similar) and polyhedrin (49-58% identical and 62-72% similar) proteins. Phylogenetic analyses based on granulin and polyhedrin genes as well as on their 5'-untranslated sequences (5'-UTSs) indicated that HbGV was more closely related to GVs isolated from the tortricids, Cryptophlebia leucotreta (ClGV), Cydia pomonella (CpGV) and Choristoneura fumiferana (CfGV) than to other GVs and NPVs. This analysis provides preliminary evidence for a correlation between GV tissue tropism and the phylogeny of lepidopteran families, suggesting that GVs attacking species of Tortricidae and Zygaenidae are ancestral to those attacking species of the family Noctudiae.

5' Untranslated Regions↗

PubMed1994

The granulin gene region of Cryptophlebia leucotreta granulosis virus: sequence analysis and phylogenetic considerations.

The nucleotide sequence of a 2.2 kb genome region of the baculovirus Cryptophlebia leucotreta granulosis virus (CIGV) encompassing the granulin gene and a second open reading frame, designated ORF909, was determined. The putative granulin ORF comprises 744 nucleotides encoding a polypeptide with a predicted M(r) of 29.3 K. The 5' leader of the granulin gene contains a canonical late baculovirus promoter (ATAAG) and differs only in two nucleotide positions from the sequence of Cydia pomonella granulosis virus (CpGV). Sequence comparison with the granulin genes of different granulosis viruses indicated a very close relationship between CIGV and CpGV of about 96% amino acid identity. ORF909 is 909 nucleotides in length and encodes a potential protein of M(r) 36.2K. It contains two zinc finger-like motifs, similar to ME53, which was previously described for Autographa californica multiple nucleocapsid nuclear polyhedrosis virus. ORF909 protein is significantly shorter than ME53 protein, lacking more than 105 amino acids of the amino terminus of ME53. Both genes contain an early and a late promoter motif suggesting a similar temporal regulation. Homologous sequences to ORF909 are also present upstream of the granulin genes of other granulosis viruses (GVs) indicating that this ORF is common in GVs.

Amino Acid Sequence↗

PubMed2005

Molecular cloning and characterization of a granulin-containing cysteine protease SPCP3 from sweet potato (Ipomoea batatas) senescent leaves.

Granulins are a family of evolutionarily ancient proteins that are involved in regulating cell growth and division in animals. In this report a full-length cDNA, SPCP3, was isolated from senescent leaves of sweet potato (Ipomoea batatas). SPCP3 contains 1389 nucleotides (462 amino acids) in its open reading frame, and exhibits high amino acid sequence homologies (ca. 64-73.6%) with several plant granulin-containing cysteine proteases, including potato, tomato, soybean, kidney bean, pea, maize, rice, cabbage, and Arabidopsis. Gene structural analysis shows that SPCP3 encodes a putative precursor protein. Via cleavage of the N-terminal propeptide, it generates a protein with 324 amino acids (from the 139th to the 462nd amino acid residues), which contains two main domains: the conserved catalytic domain with the putative catalytic residues (the 163rd Cys, 299th His and 319th Asn) and the C-terminal granulin domain (from the 375th to the 462nd amino acid residues). Semi-quantitative RT-PCR and protein gel blot hybridization showed that SPCP3 gene expression was enhanced significantly in natural senescent leaves and in dark- and ethephon-induced senescent leaves, but was almost undetectable in mature green leaves, veins, and roots. Phylogenic analysis showed that SPCP3 displayed close association with a group of plant granulin-containing cysteine proteases which have been implied to be involved in programmed cell death. In conclusion, sweet potato SPCP3 is a functional, senescence-associated gene. Its mRNA and protein levels were significantly enhanced in natural and induced senescing leaves. The physiological role and/or function of SPCP3 associated with programmed cell death during leaf senescence were also discussed.

Amino Acid Sequence↗