Biomedical subjects

M C Kiefer

Publications and source records attributed to M C Kiefer.

At least 37 records · Page 2Linked to original sources

PubMed1994

Characterization of the chromosomal gene and promoter for human insulin-like growth factor binding protein-5.

To better understand the regulation of insulin-like growth factor binding protein-5 (IGFBP-5) expression, we cloned the IGFBP-5 gene from human genomic libraries and identified a region in the 5' flanking sequence which functions as a promoter. The human IGFBP-5 gene is divided into four exons which, primarily due to a first intron of approximately 25 kilobases, span approximately 33 kilobases of DNA. Southern analysis identified a single copy of the IGFBP-5 gene in the haploid human genome, and several independent mapping strategies found this gene tightly linked with, and in opposite transcriptional orientation to, the IGFBP-2 gene at chromosomal region 2q33-34. Primer extension studies identified the IGFBP-5 mRNA cap site 772 base pairs (bp) 5' to the first nucleotide of the translation start codon. Analysis of the 5'-flanking sequence identified a potential TATA element beginning 33 bp 5' to the mRNA cap site. When a DNA fragment containing this cap site and 461 bp of upstream sequence was placed 5' to the chloramphenicol acetyltransferase reporter gene and transfected into MDA-MB-468 human breast cancer cells, it directed chloramphenicol acetyltransferase expression in an orientation-specific manner, suggesting that this region contains elements essential for IGFBP-5 promoter activity.

Amino Acid Sequence↗

PubMed1994

Protection from Fas-mediated apoptosis by a soluble form of the Fas molecule.

Fas is an apoptosis-signaling receptor molecule on the surface of a number of cell types. Molecular cloning and nucleotide sequence analysis revealed a human Fas messenger RNA variant capable of encoding a soluble Fas molecule lacking the transmembrane domain because of the deletion of an exon encoding this region. The expression of soluble Fas was confirmed by flow cytometry and immunocytochemical analysis. Supernatants from cells transfected with the variant messenger RNA blocked apoptosis induced by the antibody to Fas. Levels of soluble Fas were elevated in patients with systemic lupus erythematosus, and mice injected with soluble Fas displayed autoimmune features.

Amino Acid Sequence↗

PubMed1994

Regulation of insulin-like growth factor binding protein 4 by a specific insulin-like growth factor binding protein 4 proteinase in normal human osteoblast-like cells: implications in bone cell physiology.

Insulin-like growth factor binding protein 4 (IGFBP-4) is secreted by normal human osteoblast-like cells (hOB) and is a potent inhibitor of insulin-like growth factor (IGF) action in vitro. In previous studies, IGF treatment of hOB in culture led to markedly reduced medium levels of IGFBP-4 as detected by western ligand blotting. In the present study, incubation of hOB-conditioned medium (hOB-CM) with IGF under cell-free conditions resulted in a similar loss of IGFBP-4. Both IGF-I and IGF-II were capable of inducing a decrease in IGFBP-4; however, IGF-II was more effective. When the six characterized IGFBP were added to hOB-CM, only IGFBP-4 disappeared in response to IGF-II addition. This IGF-regulated loss of IGFBP-4 was inhibited by metalloproteinase inhibitors and appeared to be due to a proteinase that cleaved IGFBP-4 in 18 and 14 kD fragments identified by western immunoblotting. Conditioned media from eight of eight different donor hOB lines tested exhibited IGFBP-4 proteinase activity. To assess the biologic consequences of IGF-II-induced IGFBP-4 proteolysis, we treated hOB with IGF-II for 5 h, which decreased medium IGF-BP-4 by 70%, and then measured IGF-I and insulin stimulation of [3H]thymidine incorporation. IGF-II itself was not mitogenic and had no effect on insulin-stimulated [3H]thymidine incorporation. However, pretreatment of cultured hOB with IGF-II enhanced IGF-I-stimulated [3H]thymidine incorporation threefold.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1994

Role of muscle insulin-like growth factors in nerve sprouting: suppression of terminal sprouting in paralyzed muscle by IGF-binding protein 4.

The protracted absence of muscle activation initiates complex cellular and molecular reactions aimed at restoring functional neuromuscular transmission and preventing degenerative processes. A central aspect of these reactions is the sprouting of intramuscular nerves in the vicinity of inactivated muscle fibers. Sprouts emerging from terminal nerve branches and nodes of Ranvier can reestablish functional contacts with inactive muscle fibers, and this is an essential restorative process in pathological conditions of the neuromuscular system. Due to their rapid upregulation in inactive skeletal muscle fibers and their ability to induce nerve sprouting in adult muscle, insulin-like growth factors (IGFs) are candidate signaling molecules to promote restorative reactions in the neuromuscular system. In this study we have exploited the high affinity and specificity of IGF-binding protein 4 (IGF-BP4) and IGF-BP5 for IGF1 and IGF2 to determine whether these growth factors are involved in the nerve sprouting reaction in paralyzed skeletal muscle. In tissue culture experiments with sensory- and motoneurons we demonstrate that the neurite promoting activity of IGF1 is blocked by IGF-BP4, and that a similar IGF-BP-sensitive activity is detected in muscle extracts from paralyzed, but not from control muscle. In in vivo experiments, we show that local delivery of IGF-BP4 to Botulinum toxin A-paralyzed skeletal muscle effectively prevents nerve sprouting in that muscle. Our findings indicate that muscle IGFs play an essential role in intramuscular nerve sprouting. In addition, these findings suggest that IGFs are major signaling factors from inactivated muscle to promote local restorative reactions, including interstitial cell proliferation and nerve sprouting.

Animals↗

PubMed1993

Carboxy-truncated insulin-like growth factor binding protein-5 stimulates mitogenesis in osteoblast-like cells.

Recently we demonstrated that a 23 kDa form of IGFBP-5, derived from osteoblast-like cells, stimulates osteoblast mitogenesis and enhances IGF-I action. Because osteoblast-derived IGFBP-5 is smaller than recombinant intact IGFBP-5 (23 vs 30 kDa) and has decreased binding affinity for IGF-I, we proposed that the native 23 kDa form of IGFBP-5 was truncated at a carboxy-terminal position. We now show that a recombinant form of carboxy-truncated IGFBP-5 binds IGF-I with reduced affinity and stimulates mitogenesis in mouse osteoblasts. We also show that 125I-truncated IGFBP-5 specifically binds to osteoblast monolayers with low binding affinity, similar to that seen with native 23 kDa IGFBP-5. These data indicate that carboxy-truncated IGFBP-5 stimulates osteoblast mitogenesis and suggest that reduced IGF-binding and cell-surface attachment are local mediators of this response.

Animals↗

PubMed1993

Structure and biological activities of a heparin-derived hexasaccharide with high affinity for basic fibroblast growth factor.

We demonstrated previously that heparin-derived hexasaccharides are the smallest fragments of the polysaccharide with comparable basic fibroblast growth factor (bFGF)-modulating activity in vitro (Ishihara, M., Tyrrell, D.J., Stauber, G.B., Brown, S., Cousens, L., and Stack, R.J. (1993) J. Biol. Chem. 268, 4675-4683. In this report, a specific hexasaccharide having high affinity for recombinant human bFGF was isolated and its structure deduced by analysis of its reduced disaccharide products after treatment with nitrous acid at pH 1.5, and by 1H NMR spectroscopy. The hexasaccharide has the structure [IdoA(2-OSO3)alpha 1-4GlcNSO3(6-OSO3)alpha 1-4]2IdoA(2-OSO3)alpha 1-4 AManR(6-OSO3). The hexasaccharide effectively inhibits the binding of syndecan-transfected RO-12 UC cells to bFGF-coated wells (Ishihara, M., Tyrrell, D.J., Kiefer, M.C., Barr, P.J., and Swiedler, S.J. (1992) Anal. Biochem. 202, 310-315), prevents the binding of 125I-bFGF to confluent monolayers of adrenocortical endothelial (ACE) cells, and inhibits the bFGF-dependent proliferation of ACE cells. Unlike the heparin from which it was derived, however, the hexasaccharide cannot promote the binding of 125I-bFGF to a recombinant high affinity bFGF receptor (flg) or restore the bFGF-dependent proliferative response to ACE cells grown in the presence of 5 mM sodium chlorate. Collectively, these data indicate that a hexasaccharide can be as effective as heparin as an antagonist of bFGF-mediated cell mitogenesis.

Animals↗

PubMed1993

Posttranslational regulation of insulin-like growth factor binding protein-4 in normal and transformed human fibroblasts. Insulin-like growth factor dependence and biological studies.

Insulin-like growth factor binding protein-4 (IGFBP-4) is a 24-26-kD protein expressed by a variety of cell types in vivo and in vitro. Treatment of normal adult human fibroblasts with 10 nM insulin-like growth factor II (IGF-II) for 24 h resulted in an 85% decrease in endogenous IGFBP-4, as assessed by Western ligand blot analysis of the conditioned medium. Incubation of human fibroblast-conditioned medium (HFCM) with IGF-II under cell-free conditions led to a similar loss of IGFBP-4. This posttranslationally regulated decrease in IGFBP-4 appeared to be due to a protease in HFCM: (a) It could be prevented with specific protease inhibitors or incubation at 4 degrees C; (b) proteolysis of recombinant human (rh) IGFBP-4 required HFCM; (c) immunoblotting and radiolabeling confirmed cleavage of IGFBP-4 into 18- and 14-kD IGFBP-4 fragments. The protease was specific for IGFBP-4, and was strictly dependent on IGFs for activation. IGF-II was the most effective of the natural and mutant IGFs tested, inducing complete hydrolysis of rhIGFBP-4 at a molar ratio of 0.25:1 (IGF/IGFBP-4). Simian virus 40-transformed adult human fibroblasts also expressed IGFBP-4 and IGFBP-4 protease, as well as an inhibitor of IGFBP-4 proteolysis. In biological studies, intact rhIGFBP-4 potently inhibited IGF-I-stimulated [3H]aminoisobutyric acid uptake, whereas proteolyzed rhIGFBP-4 had no inhibitory effect. In conclusion, these data provide evidence for a novel IGF-dependent IGFBP-4-specific protease that modifies IGFBP-4 structure and function, and indicate a preferential role for IGF-II in its activation. Posttranslational regulation of IGFBP-4 may provide a means for cooperative control of local cell growth by IGF-I and IGF-II.

Adult↗

PubMed1993

Rat ovarian insulin-like growth factor-binding protein-6: a hormonally regulated theca-interstitial-selective species with limited antigonadotropic activity.

Most but not all of the established insulin-like growth factor-binding proteins (IGFBPs) are expressed in the rat ovary. To continue the process of characterizing these ovarian IGFBPs, a solution hybridization/RNase protection assay was used to study IGFBP-6 gene expression, cellular localization, and hormonal regulation in the immature rat ovary. Total RNA was hybridized with a 458-base long 32P-labeled rat IGFBP-6 cRNA. A single protected fragment (380 bases long) corresponding to IGFBP-6 transcripts was identified in RNA from ovary and lung, but not kidney or liver. The amount of IGFBP-6 transcripts was higher in ovaries from immature rats (25-28 days old) than in ovaries from adult rats and was higher in theca-interstitial than in granulosa cell preparations. Hypophysectomy resulted in a significant (P < 0.05) 2.3 +/- 0.7-fold (mean +/- SD) increase in whole ovarian IGFBP-6 transcripts. This suggests that ovarian IGFBP-6 gene expression is subject to inhibition by one or more pituitary hormones. Therefore, the effect of replacement of FSH, GH, diethylstilbestrol (DES), or combinations thereof was evaluated. Treatment with FSH resulted in a 2.4-fold decrease (P < 0.05) in the abundance of ovarian IGFBP-6 transcripts relative to that in hypophysectomized controls. Provision of DES yielded comparable results. Moreover, the combined provision of FSH and DES resulted in a synergistic decrease (6.0-fold) in ovarian IGFBP-6 transcripts. In contrast, treatment of hypophysectomized rats with GH proved without effect. However, treatment with FSH or DES in addition to GH resulted in a decrease in ovarian IGFBP-6 transcripts (3.9- and 2.7-fold, respectively). To assess the role of ovarian IGFBP-6, its influence on gonadotropin action in primary cultures of rat granulosa cells was also studied. Increasing concentrations (0.01-1 micrograms/ml) of recombinantly expressed human IGFBP-6 did not inhibit the FSH-supported accumulation of either progesterone or estradiol. These findings 1) establish the theca-interstitial compartment of the immature rat ovary as a prominent site of IGFBP-6 gene expression, 2) implicate FSH and DES as inhibitors of IGFBP-6 transcript levels in the whole ovary, 3) and disclose the limited antigonadotropic action of IGBP-6F on the rat granulosa cell.

Animals↗

PubMed1993

Regulation and biological effect of endogenous insulin-like growth factor binding protein-5 in human osteoblastic cells.

U-2 human osteosarcoma cells secrete a 29/32/34 kilodalton (kDa) insulin-like growth factor binding protein (IGFBP) identified as O-glycosylated IGFBP-5. Treatment of U-2 cells with IGF-I markedly increased medium levels of IGFBP-5 in a concentration- and time-dependent manner; other skeletal regulatory factors (GH, insulin, PTH, dexamethasone, beta-estradiol, 1,25-dihydroxyvitamin D3, transforming growth factor-beta) had no effect. IGF-I increased IGFBP-5 levels in the culture medium 10-fold without influencing IGFBP-5 messenger RNA abundance. IGF-I, IGF-II, and the IGF-I analog [1-27Gly(4)38-70] IGF-I bound IGFBP-5 with high affinity and, when added to U-2 cultures, effectively promoted IGFBP-5 accumulation in the medium. On the other hand, des(1-3)IGF-I and [QAYL]IGF-I, IGF-I analogs that did not bind IGFBP-5, failed to elicit an increase in medium IGFBP-5. Cell-free incubation of recombinant human (rh) IGFBP-5 in U-2 conditioned medium resulted in a marked reduction of detectable rhIGFBP-5; the presence of IGF-I or IGF-II peptide partially prevented this decrease. By immunoblot analysis, loss of intact rhIGFBP-5 (29-kDa unreduced, 34-kDa reduced) coincided with the appearance of a 16-kDa proteolytic fragment. U-2 conditioned medium contained immunoreactive IGFBP-5 at 29-34-kDa, 20-kDa, 17-kDa, and 16-kDa. Endogenous IGFBP-5 inhibited IGF-I but not des(1-3)IGF-I-stimulated U-2 cell proliferation. In conclusion, IGF peptides can regulate the availability of IGFBP-5 in osteoblast-like cells by impeding IGFBP-5 proteolysis. The biological consequence of increased medium IGFBP-5 appears to be decreased cell responsiveness to IGF-I stimulation.

Blotting, Northern↗

PubMed1993

Recombinant human insulin-like growth factor binding proteins 4, 5, and 6: biological and physiochemical characterization.

We have recently cloned cDNAs encoding human insulin-like growth factor binding proteins (IGFBP)-4, -5 and -6 and have now expressed these BPs in yeast as ubiquitin (Ub)-IGFBP fusion proteins. Western ligand blotting with 125I-IGF II under nonreducing conditions of recombinant human (rh) IGFBP-containing yeast lysates revealed specific binding bands for IGFBP-4, -5, and -6 at apparent molecular masses of 24-26, 30-32, and 24-26 kDa, respectively, indicating expression and processing of the fusion proteins. HPLC purified rhIGFBPs had virtually the same amino acid composition, amino acid number, and NH2-terminal sequences as the native BPs. Rabbit antiserum directed against each rhIGFBP-4, -5 and -6 reacted specifically with the respective rhIGFBP as well as with the native human counterpart and displayed very low cross-reactivity with other IGFBPs. Except for the affinity of rhIGFBP-6 for IGF I (Ka = 8.5 x 10(8) M-1), the affinity constants of the three IGFBPs for IGF I and II lie between 1.7 and 3.3 x 10(10) M-1. When present in excess, rhIGFBP-4, -5, and -6 inhibited IGF I- and II-stimulated DNA and glycogen synthesis in human osteoblastic cells, although rh-IGFBP-6 had only a weak inhibitory effect on IGF I in agreement with its relatively lower IGF I affinity constant.

Amino Acid Sequence↗

PubMed1992

Selection of COS cell mutants defective in the biosynthesis of heparan sulfate proteoglycan.

A simple procedure using human basic fibroblast growth factor (FGF) was utilized for the selection of COS cell mutants with defects in the biosynthesis or expression of heparan sulfate proteoglycan (HSPG). Our approach was based on the strong binding affinity exhibited by COS cells to human basic FGF that had been adsorbed to plastic dishes. Cell binding to basic FGF could be inhibited by heparin and heparan sulfate (HS), but not by chondroitin sulfate, dermatan sulfate, keratan sulfate, or hyaluronic acid, suggesting that the cell binding involved an interaction between basic FGF and cell surface heparin-like molecules. COS cells were treated with ethyl methanesulfonate and four stable mutants were subsequently isolated that did not bind strongly to basic FGF adsorbed to plastic. These mutants cell lines (CM-2, CM-8, CM-9, and CM-15) exhibited significantly reduced 35SO4 incorporation into HS (40-70% depending on the cellular pool analyzed). In one of these cell lines, CM-15, the incorporation of [6-3H]glucosamine into HS was unaltered, suggesting that the extent of oligosaccharide polymerization was equivalent to that observed for the wild-type cells. Structural analysis revealed that N-sulfated glucosamine residues were present much less frequently in HS derived from these cells as compared with that derived from wild-type cells. Furthermore, CM-15 was found to be three-fold deficient in HS N-sulfotransferase activity, but contained wild-type levels of HS O-sulfotransferase activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Characterization of recombinant human insulin-like growth factor binding proteins 4, 5, and 6 produced in yeast.

The insulin-like growth factor binding protein (IGFBP) family comprises six structurally distinct, but highly homologous proteins. They have been identified in serum and other biological fluids, tissue extracts, and cell culture media. We have recently cloned cDNAs encoding human IGFBP-4, -5, and -6 and have now expressed these BPs in yeast as ubiquitin (Ub)-IGFBP fusion proteins. Western ligand blotting with 125I-IGF II under nonreducing conditions of recombinant human (rh) IGFBP-containing yeast lysates revealed specific binding bands for IGFBP-4, -5, and -6 at apparent molecular masses of 24-26, 30-32, and 24-26 kDa, respectively, indicating processing of the fusion proteins. High-performance liquid chromatography-purified rhIGFBPs had virually the same amino acid composition, amino acid number, and NH2-terminal sequences as the native BPs. Except for the affinity of rhIGFBP-6 for IGF I (Ka = 8.5 x 10(8) M-1), the affinity constants of the three IGFBPs for IGF I and II lie between 1.7 and 3.3 x 10(10) M-1, i.e. 25-100 times higher than the IGF I and II affinities of the type I IGF receptor. When present in excess, rhIGFBP-4, -5, and -6 inhibited IGF I- and II-stimulated DNA and glycogen synthesis in human osteoblastic cells, but rhIGFBP-6 had only a weak inhibitory effect on IGF I in agreement with its relatively lower IGF I affinity constant. The results of this study show that the primary effect of the three rhIGFBPs is the attenuation of IGF activity and suggest that IGFBPs contribute to the control of IGF-mediated cell growth and metabolism.

Amino Acid Sequence↗

PubMed1992

A cell-based assay for evaluating the interaction of heparin-like molecules and basic fibroblast growth factor.

A simple panning procedure that allows for the evaluation of interactions between various heparin-like molecules and basic FGF has been developed. This assay measures the ability of compounds to inhibit the interaction of transfected human lymphoblastoid cells, UC 729-6 (UC cells), expressing hamster syndecan and basic FGF-coated plastic plates. The transfected cells bind rapidly to basic FGF-coated plates while the control cells do not bind well. Binding of the transfected cells to basic FGF was inhibited by heparin and heparin sulfate (HS), but not by chondroitin sulfate, dermatan sulfate, keratan sulfate, and hyaluronic acid. There was little inhibition of binding by chemically modified heparin such as completely desulfated, N-acetylated heparin, completely desulfated, N-sulfated heparin, and N-desulfated, N-acetylated heparin. These results suggested that both the N-sulfate and O-sulfate groups of heparin are required for binding to basic FGF. In addition, inhibition by oligosaccharides derived from depolymerized heparin increased with fragment size; partial inhibition was observed with oligosaccharides as small as hexamers. The biochemical basis for the binding of transfected cells to basic FGF was established by showing a significant increase of 35SO4 incorporation into HS. In particular, the level of 35SO4-HS in the trypsin-releasable (cell surface) pool increased fivefold. This increase was accounted for by demonstration of the presence of HS on immunoprecipitated syndecan from the transfected cells.

Animals↗

PubMed1992

Structure and expression of the gene for Pv200, a major blood-stage surface antigen of Plasmodium vivax.

Molecular cloning and structure analysis of the gene encoding the Pv200 protein of the Sal-1 strain of Plasmodium vivax revealed an overall identity of 34-37% when the deduced amino acid sequence was compared with the sequences of various major merozoite surface antigens of Plasmodium falciparum, Plasmodium yoelii and Plasmodium chabaudi. When the Sal-1 Pv200 sequence was compared with the corresponding sequence from the Belèm strain of P. vivax, it was found that the two merozoite surface antigens were relatively well conserved with an overall amino acid sequence identity of 81%. A region of 23 repeated glutamine residues, found in the sequence of the Belèm isolate was not found, however, in the Sal-1 sequence. Amino- and carboxy-terminal domains of the Pv200 protein were expressed in the yeast Saccharomyces cerevisiae. Each recombinant protein was shown to react with antibodies in sera from splenectomized Bolivian Saimiri monkeys that had been infected previously with P. vivax, and in human sera from individuals with a history of exposure to vivax malaria. The availability of recombinant DNA-derived Pv200 proteins will now allow a full assessment of their utility in the diagnosis and immunoprophylaxis of the benign tertian malaria associated with P. vivax infection.

Amino Acid Sequence↗

PubMed1991

Identification and molecular cloning of two new 30-kDa insulin-like growth factor binding proteins isolated from adult human serum.

Three insulin-like growth factor binding proteins (IGFBP) with apparent molecular masses of 24, 28-30, and 30 kDa, nonreduced, have been isolated from human serum. The 15 NH2-terminal amino acids of the 24-kDa binding protein are identical with those of the 30-kDa BP. The apparent molecular mass of the latter is reduced to 24 kDa by N-glycanase, suggesting that the 30-kDa BP is the glycosylated form of the isolated 24-kDa BP. The complete amino acid sequences derived from the cloned cDNAs represent two new IGFBPs. They are tentatively termed IGFBP-4 and -5. The prepeptide sequences of BP-4 and -5 contain 27 and 21, the mature proteins 213 and 237 amino acids, respectively (Mr = 22,610 and 25,980). The NH2- and COOH-terminal thirds of BP-4 and -5 display pronounced homology to the other three human BPs. 16 of the 16-20 cysteines and 37 of the 213-289 amino acids (12.8-17.1%) are conserved in all five mature BPs. 10 amino acid positions located in the NH2-terminal region and shared by BP-1, -2, -3, and -5 are different in BP-4. These differences may account for the preferential affinity of BP-4 for IGF II. A most intriguing homology exists between the COOH-terminal quarter of the five IGFBPs, 10 repetitive domains of human thyroglobulin, a gastrointestinal tumor-associated antigen, and the invariant chain of the class II histocompatibility antigen. The cDNAs of five human IGFBPs are now available. They will allow their expression and production in sufficient quantities for in vivo studies to unravel their role in growth and metabolism.

Amino Acid Sequence↗

PubMed1991

Molecular cloning of a new human insulin-like growth factor binding protein.

We have cloned a new insulin-like growth factor's binding protein (IGFBP) from a human osteosarcoma cDNA library. Two conserved regions in the COOH-terminal third of the five known human IGFBPs were used to design primers and to perform polymerase chain reaction (PCR) with osteosarcoma cDNA as a template. One of the eight PCR products encoded a unique IGFBP sequence. The DNA sequence was used to synthesize probes to screen an osteosarcoma cDNA library and isolate full length cDNA clones. The amino acid sequence was deduced from one of them. It contains two possible signal peptidase cleavage sites yielding a mature molecule of 257 or 252 amino acids, and 18 cysteines in identical positions to the other IGFBPs. The most pronounced homology exists with human IGFBP-3 (50% in the NH2- and 45% in the COOH-terminal region).

Amino Acid Sequence↗

PubMed1991

cDNA and gene structure for a human subtilisin-like protease with cleavage specificity for paired basic amino acid residues.

A cDNA encoding the human fur gene product was isolated from a human hepatoma cell line. The cDNA encodes a protein with significant amino acid sequence identity to the prokaryotic subtilisin family of serine proteases. More extensive sequence identity was found when the protein was compared with eukaryotic proteases such as PRB1 of Saccharomyces cerevisiae, and with PC2 and PC3, the only other known mammalian subtilisin-like proteases. In contrast to these proteins, however, the fur gene product shares a more extensive topographic and functional homology with the KEX2 endoprotease of S. cerevisiae. Each protease contains a signal peptide, a glycosylated extra cytoplasmic domain, a hydrophobic membrane-spanning region, and a short, hydrophilic "tail" sequence. As with KEX2, the expressed human protease was shown to cleave mammalian proproteins at their paired basic amino acid processing sites. We have, therefore, proposed the function-based acronym PACE (paired basic amino acid cleaving enzyme) for this prototypic mammalian proprotein processing enzyme.

Amino Acid Sequence↗

PubMed1991

Identification of a second human subtilisin-like protease gene in the fes/fps region of chromosome 15.

A cDNA encoding a novel human subtilisin-like protease was identified by a polymerase chain reaction (PCR) methodology. PCR primers were designed to be specific for the subfamily of eukaryotic subtilisin-like proteases with specificity for paried basic amino acid residue processing motifs. The gene encoding this protease, designated PACE4, also encoded a smaller subtilisin-related polypeptide derived by alternate mRNA splicing. The deduced PACE4 protein sequence contained a number of interesting features not present in other family members, including an extended signal peptide region, and a relatively large carboxyl-terminal cysteine-rich region with no obvious membrane anchor sequence. As with the fur gene product, the tissue distribution of PACE4 was widespread, with comparatively higher levels in the liver. An additional relationship to the fur gene product was shown by chromosomal localization studies. The close proximity of the fur and PACE4 genes on chromosome 15 suggests that these genes probably evolved from a common ancestor by gene duplication.

Amino Acid Sequence↗