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A Rehemtulla

Publications and source records attributed to A Rehemtulla.

At least 55 records · Page 3Linked to original sources
PubMed1995

Proteolytic maturation of protein C upon engineering the mouse mammary gland to express furin.

Endoproteolytic processing of the human protein C (HPC) precursor to its mature form involves cleavage of the propeptide after amino acids Lys-2-Arg-1 and removal of a Lys156-Arg157 dipeptide connecting the light and heavy chains. This processing was inefficient in the mammary gland of transgenic mice and pigs. We hypothesized that the protein processing capacity of specific animal organs may be improved by the coexpression of selected processing enzymes. We tested this by targeting expression of the human proprotein processing enzyme, named paired basic amino acid cleaving enzyme (PACE)/furin, or an enzymatically inactive mutant, PACEM, to the mouse mammary gland. In contrast to mice expressing HPC alone, or to HPC/PACEM bigenic mice, coexpression of PACE with HPC resulted in efficient conversion of the precursor to mature protein, with cleavage at the appropriate sites. These results suggest the involvement of PACE in the processing of HPC in vivo and represent an example of the engineering of animal organs into bioreactors with enhanced protein processing capacity.

Amino Acid Sequence↗
PubMed1993

Cloning and functional expression of a cDNA encoding the catalytic subunit of bovine enterokinase.

Enterokinase (enteropeptidase) is a heterodimeric serine protease that is responsible for the physiological activation of trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the sequence (Asp)4-Lys. In this paper, we report the cloning and functional expression of a cDNA encoding the catalytic domain (light chain) of bovine enterokinase. The nucleotide sequence of this cloned cDNA predicts a 235-amino acid polypeptide that shares a high degree of homology with a variety of mammalian serine proteases involved in digestion, coagulation, and fibrinolysis. We have developed a novel expression method for the enzyme which utilizes the secretory leader and propeptide of the mammalian serine protease PACE fused to the enterokinase light chain amino terminus. Efficient cleavage of the paired dibasic amino acid cleaving enzyme (PACE) propeptide was achieved by coexpression with human PACE or yeast KEX2. The mature product migrates at 43,000 Da on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, comparable to light chain derived from bovine duodena, and exhibited high levels of activity in cleaving the enterokinase-specific fluorogenic substrate Gly-(Asp)4-Lys-beta-naphthylamide. The recombinant single-chain form of enterokinase was also capable of activating trypsinogen, indicating that the specificity of the enzyme for its natural substrate is retained even in the absence of the noncatalytic enterokinase heavy chain.

Amino Acid Sequence↗
PubMed1993

PACE4 is a member of the mammalian propeptidase family that has overlapping but not identical substrate specificity to PACE.

Proteins that transit the constitutive pathway of secretion frequently require proteolytic processing after a pair of basic amino acids to attain their full functional activity. A ubiquitously expressed calcium-dependent subtilisin-like serine protease, named PACE or furin, can cleave precursor polypeptides specifically at pairs of basic amino acids where an arginine residue is present in the P4 position. Another member of this protease family, PACE4, was cloned recently by a PCR-based strategy and was also shown to be ubiquitously expressed. We have expressed PACE4 by transient DNA transfection of COS-1 cells and have shown that the cDNA encodes a 120-kDa polypeptide that is present in cell extracts but not in conditioned medium of transfected cells. The substrate specificities of PACE and PACE4 for cleavage of pro-von Willebrand factor were studied in parallel using a transient DNA cotransfection system. Like PACE, PACE4 was able to process pro-vWF to its mature form, and efficient cleavage required both the P4 arginine and the P2 lysine. These data, taken together with previously published data showing that PACE4 cannot process pro-factor IX, demonstrate that PACE and PACE4 have overlapping but not identical substrate specificities. Further differences between PACE and PACE4 specificities were elucidated by monitoring inhibition of processing activity mediated by the serine protease inhibitor alpha 1-antitrypsin Pittsburgh mutant. Pro-vWF processing by PACE was inhibited by expression of the alpha 1-antitrypsin Pittsburgh mutant, whereas processing of pro-vWF by PACE4 was not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Expression of bovine vitamin K-dependent carboxylase activity in baculovirus-infected insect cells.

A vitamin K-dependent carboxylase has recently been purified from bovine liver microsomes and candidate cDNA clones have been isolated. Definitive identification of the carboxylase remains circumstantial since expression of candidate carboxylase cDNAs in mammalian cells is confounded by the presence of endogenous carboxylase activity. To overcome this problem, a recombinant strain of baculovirus (Autographa california nuclear polyhedrosis virus, AcMNPV) encoding a putative carboxylase (vbCbx/AcMNPV) was used to infect Sf9 insect cells, which we demonstrate have no endogenous carboxylase activity. Infection with vbCbx/AcMNPV conferred vitamin K-dependent carboxylase activity to Sf9 insect cells. Carboxylase activity was demonstrated to peak 2-3 days after infection with vbCbx/AcMNPV. Metabolic radiolabeling with L-[35S]methionine revealed that the 90-kDa recombinant protein is the major protein synthesized at the time of peak activity after infection. An anti-peptide antibody directed against residues 86-99 reacted with bovine liver carboxylase on Western blot analysis and immunoprecipitated recombinant carboxylase from infected Sf9 microsomal protein preparations. Since Sf9 insect cells lack endogenous vitamin K-dependent carboxylase activity, expression of carboxylase activity in Sf9 insect cells with recombinant baculovirus demonstrates that the protein encoded by this cDNA is a vitamin K-dependent gamma-glutamyl carboxylase.

Animals↗
PubMed1993

In vitro and in vivo functional characterization of bovine vitamin K-dependent gamma-carboxylase expressed in Chinese hamster ovary cells.

Coagulation factor IX is a serine protease for which high-level expression of biologically active protein in heterologous cells is limited due to inefficient proteolytic removal of the propeptide as well as vitamin K-dependent carboxylation of multiple amino-terminal glutamic acid residues. We have overexpressed the vitamin K-dependent gamma-carboxylase cDNA and monitored its ability to improve factor IX processing in Chinese hamster ovary (CHO) cells. From amino acid sequence analysis of bovine liver vitamin K-dependent gamma-carboxylase, degenerate oligonucleotides were used to isolate a 3.5-kbp bovine cDNA that encoded a 758-residue open reading frame. Expression of the cDNA in COS-1 and CHO cells yielded 17- and 16-fold increases in the in vitro gamma-carboxylase activity of microsomal preparations, respectively. Anti-serum raised against a predicted peptide sequence reacted with a 94-kDa polypeptide in the partially purified bovine liver preparation as well as in stably transfected CHO cells. The amount of antibody reactivity correlated with the increased ability to carboxylate a peptide substrate in vitro. These results strongly support the conclusion that the cDNA encodes the vitamin K-dependent gamma-carboxylase. Transient transfection of the gamma-carboxylase expression vector into factor IX-expressing CHO cells did not improve the specific procoagulant activity of secreted factor IX. In contrast, transfection of an expression vector encoding the propeptide processing enzyme PACE (paired basic amino acid cleaving enzyme) did improve the specific activity of secreted factor IX by 3-fold. These results demonstrate that the ability of CHO cells to modify glutamic acid residues to gamma-carboxyglutamic acid in secreted factor IX is not limited by the expression of the vitamin K-dependent gamma-carboxylase alone.

Amino Acid Sequence↗
PubMed1993

PACE/furin can process the vitamin K-dependent pro-factor IX precursor within the secretory pathway.

Factor IX is synthesized as a precursor polypeptide which requires proteolytic cleavage of the propeptide for functional activity. Expression of factor IX at high levels in Chinese hamster ovary (CHO) cells results in the secretion of a mixture of profactor IX and mature factor IX. We have studied whether the processing of profactor IX may be mediated by the recently discovered subtilisin-like serine proteases PACE/furin and/or PACE4. Co-transfection of a PACE expression vector with a profactor IX expression vector resulted in the secretion of fully processed factor IX. In contrast, co-transfection of a PACE4 expression vector with a profactor IX expression vector did not increase processing of profactor IX to the mature form. A factor IX Arg-to-Thr mutation at the P1 position (residue 39) destroyed the ability for PACE to process profactor IX. Amino-terminal sequence analysis demonstrated that processing mediated by PACE occurred at the authentic site within profactor IX. The specificity of profactor IX processing by PACE was also evaluated by transfection of a vector encoding the serine protease inhibitor alpha 1-antitrypsin. Expression of wild-type alpha 1-antitrypsin, which does not inhibit PACE, did not influence processing of profactor IX mediated by co-expression of PACE. In contrast, the alpha 1-antitrypsin Pittsburgh mutant, which inhibits PACE, inhibited profactor IX processing activity mediated by transfected PACE as well as the endogenous CHO cell propeptide processing enzyme. Pulse-chase labeling indicated that PACE processed profactor IX late within the secretory pathway, although a secreted soluble mutant PACE was also capable of processing profactor IX in the conditioned medium. The results implicate PACE as a candidate for the enzyme that processes profactor IX in vivo.

Animals↗
PubMed1992

Tissue factor residues 157-167 are required for efficient proteolytic activation of factor X and factor VII.

The cell surface receptor tissue factor (TF) initiates coagulation by supporting the proteolytic activation of factors X and IX as well as VII to active serine proteases. Architectural similarity of TF to the cytokine receptor family suggests a strand-loop-strand structure for TF residues 151-174. Site-directed Ala exchanges in the predicted surface loop demonstrated that residues Tyr157, Lys159, Ser163, Gly164, Lys165, and Lys166 are important for function. Addition of side chain atoms at the Ser162 position decreased function, whereas the Ala exchange was tolerated. The dysfunctional mutants bound VII with high affinity and fully supported the catalysis of small peptidyl substrates by the mutant TF.VIIa complex. Lys159-->Ala substitution was compatible with efficient activation of factor X, whereas the Try157-->Ala exchange and mutations in the carboxyl aspect of the predicted loop resulted in diminished activation of factor X. The specific plasma procoagulant activity of all functionally deficient mutants increased 7- to 200-fold upon the supplementation of VIIa suggesting that TF residues 157-167 also provide important interactions that accelerate the activation of VII to VIIa. These data are consistent with assignment of the TF 157-167 region as contributing to protein substrate recognition and cleavage by the TF.VIIa complex.

Amino Acid Sequence↗
PubMed1992

Regulation of PACE propeptide-processing activity: requirement for a post-endoplasmic reticulum compartment and autoproteolytic activation.

PACE (paired basic amino acid cleaving enzyme) is a subtilisin-like serine protease involved in processing of propeptides in the constitutive secretory pathway. We here demonstrate that the transmembrane and cytoplasmic domains of PACE are required for retention in the secretory pathway but not for propeptide-cleaving activity. Addition of the endoplasmic reticulum retention signal Lys-Asp-Glu-Leu (KDEL) to the carboxyl terminus of the truncated molecule resulted in intracellular retention of the protein and loss of activity, indicating that the endoplasmic reticulum is an inappropriate environment for propeptide processing. In addition, mutation of a consensus PACE cleavage site within the amino-terminal region prevented processing of PACE to a mature form and destroyed activity. These data indicate that PACE is synthesized as a proprotein which requires autoproteolytic removal of an 81-residue pro sequence for optimal activity. A mutant form of PACE that lacked the pro sequence was nonfunctional, and addition of a pro sequence from a homologous subtilisin-like serine protease, PC2, did not restore activity. By analogy to the bacterial subtilisin family, the propeptide of PACE may guide the folding of PACE into an active enzyme.

Animals↗
PubMed1992

Preferred sequence requirements for cleavage of pro-von Willebrand factor by propeptide-processing enzymes.

Maturation of pro-von Willebrand factor (vWF) to its active form requires proteolytic processing after a pair of dibasic amino acids (-LysArg-) at residue 763. By coexpression of vWF and various propeptide processing enzymes in COS-1 cells, we here demonstrate that vWF is preferentially processed by the paired dibasic amino acid-cleaving enzyme PACE (furin). Processing of vWF by the yeast homologue of PACE, Kex2, was inefficient and not specific for the authentic site. Two additional recently identified mammalian propeptide-processing enzymes PC2 and PC3 had no detectable vWF-processing activity. The inability of PC2 and PC3 to cleave vWF was apparently not due to the absence of a transmembrane domain, since deletion of the transmembrane domain from PACE resulted in a secreted form which retained its propeptide processing activity within the secretory apparatus. The inability of PC2 and PC3 to process wild-type vWF or any of the vWF mutants described suggests different members of subtilisin-related propeptide-processing enzyme family have evolved to selectively recognize and cleave specific sets of substrates. In addition to paired dibasic residues at the propeptide cleavage site, many proteins, including vWF, also contain an arginine at the P4 position. We have generated mutant vWFs with substitutions at the P2 lysine and/or the P4 arginine to investigate their significance in substrate specificity. A conservative substitution of the P4 arginine by lysine resulted in a decrease in vWF processing by PACE, as did a nonconservative substitution to alanine. Substitution of the P2 lysine to aspartic acid decreased processing and little or no processing was detected when both the P4 and P2 were mutated to lysine and aspartic acid, respectively. These data indicate that both the P4 arginine and the P2 lysine play an important role in substrate recognition by PACE.

Amino Acid Sequence↗
PubMed1992

Cofactor residues lysine 165 and 166 are critical for protein substrate recognition by the tissue factor-factor VIIa protease complex.

High affinity binding of factor VIIa (VIIa) to its cellular receptor tissue factor (TF), as well as association of factor X with phospholipid are required for optimal assembly of the extrinsic activation complex. In addition to the interactions of substrate with phospholipid and enzyme, we here provide evidence that cofactor residues Lys-165 and Lys-166 specifically contribute to the recognition of macromolecular substrate. Ala for Lys replacement in TFA165A166 was compatible with high affinity binding of VIIa when analyzed on cell surfaces as well as in the absence of phospholipid. Dissociation of TFA165A166.VIIa did not occur with a faster rate compared to TF.VIIa, further supporting unaltered VIIa binding function of TFA165A166. Cleavage of chromogenic peptidyl substrate by TFA165A166.VIIa complexes was not diminished, demonstrating that TFA165A166 supported enhancement of catalytic function of the VIIa protease domain. In contrast, factor X activation was reduced in the presence and absence of phospholipid. Further, TFA165A166 effectively competed with wild-type TF in the cleavage of factor X at limited VIIa concentrations. Selective reduction in macromolecular substrate hydrolysis combined with normal VIIa binding by TFA165A166 indicates that the cofactor TF does contribute, either directly or indirectly via specific interactions with VIIa, to factor X recognition.

Amino Acid Sequence↗
PubMed1992

The third Trp-Lys-Ser (WKS) tripeptide motif in tissue factor is associated with a function site.

The tripeptide sequence Trp-Lys-Ser (WKS) is repeated three times in the extracellular ligand binding domain of human Tissue Factor (TF). Using site-directed mutagenesis, we replaced each of the WKS motifs in human TF by Arg-Lys-Gly (RKG), the least conserved replacement for the motif found in murine TF. This substitution in the first repeat W14KS, as well as a Trp14----Arg substitution, resulted in a structurally altered protein, whereas a conservative hydrophobic Trp14----Phe substitution resulted in a functionally normal protein. This suggests that Trp14 may contribute to a hydrophobic core rather than involvement of this motif in function. Replacement of the W45KS and W158KS motifs was associated with no detectable structural alterations; however, function was diminished with the RKG replacement of the third repeat. Mutant proteins with Lys159----Ala and Tyr157----Ala substitutions exhibited loss of function, whereas Tyr156----Ala and Ser160----Ala substitutions flanking the YWK sequence resulted in functional proteins. These data demonstrate that the W158KS motif in human TF is associated with a functional site and identify Lys159 in this motif as a functionally important residue.

Amino Acid Sequence↗
PubMed1992

Protein processing within the secretory pathway.

Endoproteolytic cleavage of hormone and neuropeptide precursors, as well as many complex proteins, such as coagulation factors and viral glycoproteins, is a key process in the generation of bioactive polypeptides. These cleavages typically occur at the dibasic amino acid residues Lys-Arg or Arg-Arg. The enzymes responsible for the processing belong to a newly discovered family of serine proteases related to the bacterial subtilisins. These include PACE (furin), PC1/PC3, PC2 and PACE4, which have all been characterized functionally and structurally.

Amino Acid Sequence↗
PubMed1991

Antibody mapping of tissue factor implicates two different exon-encoded regions in function.

Tissue Factor (TF), a small transmembrane glycoprotein, is the cellular receptor for the zymogen Factor VII and the serine protease Factor VIIa (VIIa). TF provides cofactor function for VIIa in the catalytically active (TF: VIIa) binary complex. To explore the structural loci of TF that are responsible for binding of VII and VIIa, monoclonal antibodies (MAbs) and sequence-specific polyclonal antibodies to the native TF protein were analysed for inhibition of VII binding. Two independent epitopes of MAbs were localized by reciprocal competition and by binding of the MAbs to different proteolytic fragments of TF. The epitopes were also characterized in part by progressive C-terminal deletional mutation of the TF protein. Reactivity of the anti-(locus II) MAb TF9-6G4 is consistent with epitope localization in residues Thr40-Val83, encoded by exon 3. In contrast, the anti-(locus I) MAb TF9-5G9 was reactive with fragments encompassing exon 4 (Thr106-Lys165). Antibodies to linear sequences encoded by the same two exons also inhibited VII binding. These data suggest a minimum requirement for two of the four exon-encoded regions of TF for the functional integrity of this receptor cofactor with respect to ligand recognition and high-affinity binding.

Antibodies, Monoclonal↗
PubMed1991

The integrity of the cysteine 186-cysteine 209 bond of the second disulfide loop of tissue factor is required for binding of factor VII.

The structural basis of function of tissue factor (TF), the cell surface receptor and cofactor for the serine protease factor VIIa, cannot be inferred from the primary sequence. The functional significance of the two disulfide bonded loops in the surface domain of TF has been analyzed using site-directed mutagenesis to selectively preclude covalent stabilization of these loops by pairwise substitution of serine residues for cysteines. Mutant TF lacking either the amino (TFS49S57) or carboxyl (TFS186S209) disulfide bond were expressed on the surface of cells consistent with proper processing. Each reacted with a panel of monoclonal antibodies further suggesting proper global folding of the mutant proteins. TFS186S209 exhibited a selective decrease in reactivity with an antibody directed against one epitope locus in the carboxyl aspect of the surface domain of TF. Whereas TFS49S57 was functionally comparable to the wild type protein, TFS186S209 was functionally 30-40-fold less effective, and the affinity of factor VIIa binding to this mutant was indirectly estimated to be diminished 20-fold. These data suggest that the Cys186-Cys209 disulfide bond is required to maintain conformation and implicate the disulfide loop or adjacent structures in the carboxyl half of the surface domain of TF in receptor function.

Animals↗
PubMed1991

High level expression of recombinant human tissue factor in Chinese hamster ovary cells as a human thromboplastin.

Tissue factor (TF) is the high affinity transmembrane receptor and cofactor for cellular initiation of the plasma coagulation protease cascades by factor VIIa. We describe the synthesis of recombinant huTF by stably transfected CHO cell lines carrying integrated huTF DNA, and the isolation of huTF glycoprotein with specific functional activity equivalent to natural huTF. The expression vector (pCDM8), carrying the cytomegalovirus promoter to drive transcription of a partial cDNA construct encoding the complete huTF protein chain, was cotransfected with a plasmid containing the neomycin resistance gene for selection. These clones were further selected for level of expression of huTF protein. Optimal expression compatible with stability and cell growth was approximately 13.5 x 10(6) molecules per cell. To our knowledge, this is one of the highest levels of expression described for a recombinant transmembrane receptor in mammalian cells. Recombinant huTF protein was obtained by single-step immuno-affinity purification, and exhibits heterogeneity due to N-linked glycosylation. The protein was indistinguishable from natural huTF based on functional properties of the glycoprotein reconstituted in lipid vesicles, and expression of conformational epitopes. Large scale production of recombinant huTF is feasible to permit basic studies of protein structure as well as for design of huTF thromboplastin reagents.

Animals↗
PubMed1991

Conservation of tissue factor primary sequence among three mammalian species.

Tissue factor (TF) is a transmembrane glycoprotein that serves as the cofactor for the initiation of the coagulation protease cascades. To identify conserved sequences of this molecule, a 1753-nucleotide cDNA encoding rabbit TF (rbTF) was isolated and sequenced. An open reading frame encoded a predicted precursor protein of 292 amino acids (aa), and a functionally active protein was synthesized when this cDNA was expressed in a eukaryotic cell system. The aa sequence of mature rbTF was 71% identical to human TF (huTF) and 58% to murine TF (muTF), consistent with the relative functional activity of each in human plasma. The structural organization of the protein was comparable in all three species, with a high degree of conservation of the extracellular domain, including the relative positions of cysteine residues and, to a lesser extent, the tripeptide motifs tryptophan-lysine-serine of huTF. In view of the uniform occurrence of TF functional activity throughout vertebrates, the sampling of these three distant mammalian species suggests that there is limited variance in primary sequence, consistent with the conserved function of TF.

Amino Acid Sequence↗