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Results for “Type VII secretion”

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PubMed2006

A ten-residue domain (Y11-A20) in the NH2-terminus modulates membrane association of annexin A7.

Annexin A7 (synexin, annexin VII) is postulated to promote membrane fusion during surfactant secretion in alveolar type II cells and catecholamine secretion in adrenal chromaffin cells. Recently, we demonstrated that the 1-29 residues in the NH(2)-terminus could, possibly by interaction with the COOH-terminus, influence the Ca(2+)-dependent membrane binding, aggregation, and fusion properties of annexin A7 (A7). In this study, we further investigated this 29-residue domain by evaluating several deletion and point mutations for membrane-associated functions of A7. In comparison to A7, the mutants lacking 1-29 residues (A7Delta(1-29)) or 1-21 residues (A7Delta(1-21)), but not those lacking 1-10 residues (A7Delta(1-10)) or 21-29 residues (A7Delta(21-29)), showed diminished membrane binding. Segmental deletion of 10-20 residues (A7Delta(10-20)) also decreased the protein binding to membranes. The Ca(2+)-dependent membrane aggregation of PLV with A7Delta(1-29) was maximally diminished but less so with A7Delta(10-20) or A7Delta(1-21) in comparison to that with A7. However, phospholipid vesicle (PVL) aggregation was unaffected with A7Delta(1-10) or A7Delta(21-29). The Ca(2+)-dependent membrane fusion of PLV was also diminished with A7Delta(10-20) and A7Delta(1-29), but not with A7Delta(1-10). Since the mode of annexin A7 association and function with biological membranes could be different, we also evaluated these proteins for functional changes with isolated lung lamellar bodies. In comparison to A7, the binding to lamellar bodies was diminished for A7Delta(1-29) and A7Delta(1-21) but not for A7Delta(1-10). The Ca(2+)-dependent fusion of isolated lamellar bodies with PLV was also diminished with A7Delta(1-29), but not with A7Delta(10-20) or A7Delta(1-21). Taken together, our studies suggest that the 10-residue domain (Y(11)-A(20)) in the NH(2)-terminus modifies the phospholipid binding and aggregation properties of annexin A7. For binding and fusion of biological membranes, the 10-29-residue domain may be required although the annexin A7 properties are primarily modulated through the Y(11)-A(20) domain.

Animals↗

PubMed1986

Testicular plasminogen activators during postnatal development in the rat.

In the seminiferous epithelium, Sertoli cells secrete plasminogen activator (PA) under regulation of follicle stimulating hormone, cyclic AMP and neighbouring spermatogenic cells. Recent observations suggest that preleptotene spermatocytes upon their release from the basement membrane of the seminiferous tubule are important regulators of PA secretion. To study further the role of PA's in the seminiferous tubules, we have analyzed the endogenous levels and secretion rates of PA at various ages during postnatal development, and performed biochemical analyses of the types of PA in the testis and spent media from seminiferous tubular cultures. Cyclic secretion of PA started at the age of 28 days, and from 40 days onwards, the high secretion rates were localized in stages VII and VIII of the cycle of the seminiferous epithelium. The secreted PA is most obviously of the urokinase type; both urokinase-type and tissue-type PA-like activities were found in seminiferous tubular homogenates. The increase in testicular PA levels concomitant to the onset of meiosis in the epithelium was due to the urokinase-type PA-like activity.

Aging↗

PubMed2004

A process for controlling intracellular bacterial infections induced by membrane injury.

Strategies for inhibiting phagolysosome fusion are essential for the intracellular survival and replication of many pathogens. We found that the lysosomal synaptotagmin Syt VII is required for a mechanism that promotes phagolysosomal fusion and limits the intracellular growth of pathogenic bacteria. Syt VII was required for a form of Ca2+-dependent phagolysosome fusion that is analogous to Ca2+-regulated exocytosis of lysosomes, which can be triggered by membrane injury. Bacterial type III secretion systems, which permeabilize membranes and cause Ca2+ influx in mammalian cells, promote lysosomal exocytosis and inhibit intracellular survival in Syt VII +/+ but not -/- cells. Thus, the lysosomal repair response can also protect cells against pathogens that trigger membrane permeabilization.

Animals↗

PubMed1997

Deficiency of phosphofructo-1-kinase/muscle subtype in humans impairs insulin secretion and causes insulin resistance.

Non-insulin-dependent diabetes mellitus (NIDDM) is caused by peripheral insulin resistance and impaired beta cell function. Phosphofructo-1-kinase (PFK1) is a rate-limiting enzyme in glycolysis, and its muscle subtype (PFK1-M) deficiency leads to the autosomal recessively inherited glycogenosis type VII Tarui's disease. It was evaluated whether PFK1-M deficiency leads to alterations in insulin action or secretion in humans. A core family of four members was evaluated for PFK1-M deficiency by DNA and enzyme-activity analyses. All members underwent oral and intravenous glucose tolerance tests (oGTT and ivGTT) and an insulin-sensitivity test (IST) using octreotide. Enzyme activity determinations in red blood cells showed that the father (46 yr, body mass index [BMI] 22. 4 kg/m2) and older son (19 yr, BMI 17.8 kg/m2) had a homozygous, while the mother (47 yr, BMI 28.4 kg/m2) and younger son (13 yr, BMI 16.5 kg/m2) had a heterozygous PFK1-M deficiency. DNA analyses revealed an exon 5 missense mutation causing missplicing of one allele in all four family members, and an exon 22 frameshift mutation of the other allele of the two homozygously affected individuals. The father showed impaired glucose tolerance, and the mother showed NIDDM. By ivGTT, both parents and the older son had decreased first-phase insulin secretion and a diminished glucose disappearance rate. The IST showed marked insulin resistance in both parents and the older, homozygous son, and moderate resistance in the younger son. PFK1-M deficiency causes impaired insulin secretion in response to glucose, demonstrating its participation in islet glucose metabolism, and peripheral insulin resistance. These combined metabolic sequelae of PFK-1 deficiency identify it as a candidate gene predisposing to NIDDM.

Adult↗

PubMed2000

The Tetrahymena homolog of bacterial and mammalian 4-hydroxyphenylpyruvate dioxygenases localizes to membranes of the endoplasmic reticulum.

The expression and intracellular localization of the Tetrahymena homolog of 4-hydroxyphenylpyruvate dioxygenase (HPPD) were investigated in wild-type Tetrahymena thermophila strain B1868 VII and the mutant strains IIG8, defective in food vacuole formation, MS-1, blocked in secretion of lysosomal enzymes, and SB 281, defective in mucocyst maturation. Immunoelectron microscopy and confocal laser scanning microscopy demonstrated that Tetrahymena HPPD primarily localized to membranes of the endoplasmic reticulum. In addition, Tetrahymena HPPD was detected in association with membranes of the Golgi apparatus, and transport vesicles in exponentially growing wild-type and mutant strains. In starved cells, Tetrahymena HPPD localized exclusively to membranes of small vesicles. Since no de novo synthesis of Tetrahymena HPPD takes place in cells starved for more than 30 min, these results suggest that there is a flow of Tetrahymena HPPD from the endoplasmic reticulum to small vesicles, possibly via the Golgi apparatus, and that Tetrahymena HPPD contains a signal for vesicle membrane retrieval or retention.

4-Hydroxyphenylpyruvate Dioxygenase↗

PubMedDate not supplied

Phenotype correction in murine mucopolysaccharidosis type VII by transplantation of human amniotic epithelial cells after adenovirus-mediated gene transfer.

Cell therapy with human amniotic epithelial (HAE) cells was developed as an alternative method for enzyme replacement therapy in congenital lysosomal storage disorders, but only limited therapeutic efficacy has been reported. A major drawback is insufficient production and secretion of lysosomal enzymes from HAE cells. In this study, we infected HAE cells with an E1-deleted adenoviral vector expressing human beta-glucuronidase (GUSB), and generated cells overexpressing GUSB by a hundred times as much as endogenous GUSB in untreated HAE cells. GUSB secreted from the gene-transferred HAE cells were efficiently transported to murine fibroblasts with endocytosis mediated by mannose-6-phosphate receptors. The cells were administered into the spleen of the mice with the lysosomal storage disease mucopolysaccharidosis type VII (B6/MPSVII). Approximately 10-15% of the normal GUSB activity was detected in both liver and spleen 7 days after the cell administration. Histopathological examination showed that lysosomal enlargement in tissue macrophages in the liver and the spleen had disappeared by day 14. These results suggest that transplantation of the HAE cells transduced with adenoviral vectors can be employed for the treatment of congenital lysosomal storage disorders.

Adenoviridae↗

PubMed2004

Synaptotagmin VII is targeted to dense-core vesicles and regulates their Ca2+ -dependent exocytosis in PC12 cells.

It has recently been proposed that synaptotagmin (Syt) VII functions as a plasma membrane Ca2+ sensor for dense-core vesicle exocytosis in PC12 cells based on the results of transient overexpression studies using green fluorescent protein (GFP)-tagged Syt VII; however, the precise subcellular localization of Syt VII is still a matter of controversy (plasma membrane versus secretory granules). In this study we established a PC12 cell line "stably expressing" the Syt VII-GFP molecule and demonstrated by immunocytochemical and immunoelectron microscopic analyses that the Syt VII-GFP protein is localized on dense-core vesicles as well as in other intracellular membranous structures, such as the trans-Golgi network and lysosomes. Syt VII-GFP forms a complex with endogenous Syts I and IX, but not with Syt IV, and it colocalize well with Syts I and IX in the cellular processes (where dense-core vesicles are accumulated) in the PC12 cell line. We further demonstrated by an N-terminal antibody-uptake experiment that Syt VII-GFP-containing dense-core vesicles undergo Ca2+ -dependent exocytosis, the same as endogenous Syt IX-containing vesicles. Moreover, silencing of Syt VII-GFP with specific small interfering RNA dramatically reduced high KCl-dependent neuropeptide Y secretion from the stable PC12 cell line (approximately 60% of the control cells), whereas the same small interfering RNA had little effect on neuropeptide Y secretion from the wild-type PC12 cells (approximately 85-90% of the control cells), indicating that the level of endogenous expression of Syt VII molecules must be low. Our results indicate that the targeting of Syt VII-GFP molecules to specific membrane compartment(s) is affected by the transfection method (transient expression versus stable expression) and suggested that Syt VII molecule on dense-core vesicles functions as a vesicular Ca2+ sensor for exocytosis in endocrine cells.

Animals↗

PubMed2006

Skin basement membrane and extracellular matrix proteins characterization and quantification by real time RT-PCR.

Three-dimensional gelatin-chondroitin 6 sulphate-hyanuronic acid (gelatin-C6S-HA) biomatrices were used as the scaffold to investigate the phenotypic and molecular expression of basement membrane (BM) and extracellular matrix (ECM) proteins in vitro. The cells were cultured in three different culture conditions: keratinocytes (K) monoculture, or dermal fibroblasts (FB) monoculture, or organotypic keratinocytes and dermal fibroblasts (K&FB) coculture model. The deposition of BM proteins and ECM proteins secreted by these two kinds of cells was quantitatively characterized by real time RT-PCR and examined by immunohistochemistry. The results showed that K expressed specific keratin and E-cadherin proteins, while type I collagen was secreted by FB. FB were shown to synthesize and deposit laminin 5, type IV collagen, and type VII collagen, whereas K dominantly produced integrin alpha 6 and integrin beta 4 as well as laminin 5. Interestingly, the integrin beta 4 was expressed neither in K monoculture nor in FB monoculture, but was seen in organotypic K&FB coculture model in the early culture stage. The histology studies revealed numerous features of epidermalization including a well organized basal layer of distinct cylindrical cells, granular and a horny layer, as well as complete BM formation. These results indicated that K and FB not only kept their phenotype when culturing on 3D scaffold, but also worked together to reconstruct dermal-epidermal basement membrane zone. In brief, our results directly provide the quantification in the expression of BM and ECM proteins by using real time RT-PCR in mRNA level and morphological appearance by immunostain in protein level.

Basement Membrane↗

PubMed2000

Treatment of a lysosomal storage disease, mucopolysaccharidosis VII, with microencapsulated recombinant cells.

Most lysosomal enzyme deficiencies are catastrophic illnesses with no generally available treatments. We have used the beta-glucuronidase-deficient mouse model of mucopolysaccharidosis type VII (MPS VII) to develop an alternative approach to therapy. A "universal" cell line engineered to secrete the missing enzyme is implanted in all recipients requiring the same enzyme replacement. The cells, although nonautologous, are rendered immunologically tolerant by encapsulation in microcapsules that provide protection from immune mediators. Using this strategy, we injected beta-glucuronidase-secreting fibroblasts enclosed in alginate microcapsules into mutant MPS VII mice. After 24 hr, beta-glucuronidase activity was detected in the plasma, reaching 66% of physiological levels by 2 weeks postimplantation. Significant beta-glucuronidase activity was detected in liver and spleen for the duration of the 8-week experiment. Concomitantly, the intralysosomal accumulation of undegraded glycosaminoglycans was dramatically reduced in liver and spleen tissue sections and urinary glycosaminoglycan content was reduced to normal levels. Elevated secondary lysosomal enzymes beta-hexosaminidase and alpha-galactosidase were also reduced. However, implanted mutant MPS VII mice developed antibodies against the murine beta-glucuronidase, demonstrating a potential obstacle in patients with a null mutation who react against the replaced enzyme as a foreign antigen. The antibody response was transiently circumvented with a single treatment of purified anti-CD4 antibody coadministered with the microcapsules. This resulted in increased levels and duration of beta-glucuronidase delivery. Similarly, treated heterozygous mice maintained elevated levels of beta-glucuronidase and did not develop antibodies. This novel cell-based therapy demonstrates a potentially cost-effective and nonviral treatment applicable to all lysosomal storage diseases.

Alleles↗

PubMed1988

Differentiation of fibroblast stem cells.

Primary human skin fibroblasts derived from the abdomen of 45 female donors of the four age groups 1-20, 20-40, 40-60, and 60-80 years were studied in primary explant, in primary low-density mass cultures, and in primary clonal populations in vitro. As a function of the age of the donor, primary mitotic and postmitotic fibroblasts in the three primary cell systems analysed represent heterogeneous populations with reproducible changes in the proportions of the mitotic fibroblasts MF I, MF II, MF III, and postmitotic fibroblasts PMF IV, PMF V, PMF VI, and PMF VII. These findings make it very likely that equivalent cell types exist in the connective tissue of skin in vivo, and that these cells undergo reproducible changes in the proportions of the mitotic and postmitotic counterparts in vivo as a function of the age of the donor. Secondary mitotic human skin fibroblast populations of the cell line HH-8 in vitro underwent 53.6 +/- 6.0 cumulative population doublings (CPD) in 302 +/- 27 days. If appropriate methods are applied, mitotic fibroblasts differentiate spontaneously into postmitotic fibroblasts which are kept in stationary cultures for up to 305 +/- 41 additional days. As a function of the CPD level and of the duration of stationary culture, secondary mitotic and postmitotic fibroblast populations are heterogeneous populations with reproducible changes in the proportions of mitotic fibroblasts MF I, MF II, and MF III, and postmitotic fibroblasts PMF IV, PMF V, PMF VI, and PMF VII. The seven secondary fibroblast cell types show differentiation-dependent and cell-type specific patterns of [35S]methionine polypeptides in total soluble cytoplasmic and nuclear proteins, in secreted proteins, and in membrane bound proteins. These findings make it very likely that the morphologically recognizable primary and secondary fibroblasts differentiate spontaneously along a seven stage terminal cell lineage MF I - MF II - MF III - PMF IV - PMF V - PMF VI - PMF VII in three compartments of the fibroblasts stem cell system.

Adolescent↗

PubMed2003

Injection of genetically engineered fibroblasts corrects regenerated human epidermolysis bullosa skin tissue.

Current therapeutic strategies for genetic skin disorders rely on the complex process of grafting genetically engineered tissue to recipient wound beds. Because fibroblasts synthesize and secrete extracellular matrix, we explored their utility in recessive dystrophic epidermolysis bullosa (RDEB), a blistering disease due to defective extracellular type VII collagen. Intradermal injection of RDEB fibroblasts overexpressing type VII collagen into intact RDEB skin stably restored correctly localized type VII collagen expression in vivo and normalized hallmark RDEB disease features, including subepidermal blistering and anchoring fibril defects. This article was published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.

Animals↗

PubMed2000

Somatic gene therapy for a neurodegenerative disease using microencapsulated recombinant cells.

Neurodegenerative diseases caused by lysosomal enzyme deficiencies are catastrophic illnesses with both peripheral organ and central nervous system abnormalities. The mucopolysaccharidosis type VII mouse with beta-glucuronidase deficiency was used to develop an alternate approach to gene therapy, in which a "universal" cell line engineered to secrete the missing enzyme is implanted directly into all recipients requiring the same enzyme replacement. The cells, though nonautologous, were rendered immunologically tolerable by protection in immunoisolating microcapsules. Since the blood-brain barrier impedes the passage of large molecules such as beta-glucuronidase, encapsulated cells producing beta-glucuronidase were introduced directly into the lateral ventricles of the brain. Based on this strategy, beta-glucuronidase was delivered throughout most of the central nervous system, reversing the histological pathology and reducing the previously elevated levels of lysosomal enzymes beta-hexosaminidase and alpha-galactosidase. The effectiveness of this approach was further demonstrated with improvements in the mutant circadian rhythm behavioral abnormalities. Compared to wild-type and heterozygous mice, the mutant mice had an unstable periodicity, fragmented activity, and a sixfold reduction in wheel running activity. After treatment, the mutant behavioral abnormalities were significantly improved with a more stable periodicity and a less fragmented pattern of activity. While the overall total activity level did not increase in the treated mutants, it did not show the deterioration observed in the sham-treated as well as in the untreated mutant mice. Hence, this alternative cell-based gene therapy demonstrates biochemical, histological, and behavioral efficacy and provides a potentially cost-effective and nonviral treatment applicable to all lysosomal storage diseases with neurological deficits.

Alginates↗

PubMed1999

Deficiency of phosphofructo-1-kinase/muscle subtype in humans is associated with impairment of insulin secretory oscillations.

In healthy humans, insulin is secreted in an oscillatory manner. While the underlying mechanisms generating these oscillations are not fully established, increasing evidence suggests a central role for phosphofructo-1-kinase/muscle subtype (PFK1-M), which also serves as the predominantly active PFK1 subtype in the pancreatic beta-cell. The fact that normal oscillatory secretion is impaired in subjects with impaired glucose tolerance and healthy relatives of patients with type 2 diabetes suggests that this defect may be involved in the secretory dysfunction. To evaluate a possible link between inherited PFK1-M deficiency in humans (Tarui's disease or glycogenosis type VII) and altered insulin oscillations, in vivo studies were performed. We determined basal insulin oscillations during 2 h of frequent plasma sampling in two related teen-aged individuals with homozygous and heterozygous PFK1-M deficiency compared with nondeficient, unrelated control subjects. As predicted by the underlying hypothesis, normal oscillations in insulin secretion were completely abolished in the individual with homozygous deficiency of PFK1-M and significantly impaired in the heterozygous individual, as shown by spectral density and autocorrelation analyses. Thus, deficiency of PFK1-M subtype in humans appears to be associated with an impaired oscillatory insulin secretion pattern and may contribute to the commonly observed secretion defects occurring in type 2 diabetes.

Adult↗

PubMed1984

Cellular regulation of plasminogen activator secretion during spermatogenesis.

The secretion of plasminogen activator (PA) has been found to be highly stage-specific during rat spermatogenesis. It is maximal in Stages VII and VIII of the cycle. At these stages, seminiferous tubules contain primitive type A1 spermatogonia, preleptotene and midpachytene primary spermatocytes, round and maturation-phase spermatids and Sertoli cells. The last cell types are the most likely sources of PA. To investigate which cell type might be involved in the regulation of PA secretion, we have sequentially isolated 1-mm segments of rat seminiferous tubules from Stages VI-IX with transillumination-assisted microdissection and measured PA secretion using 125I-labeled fibrinogen as substrate. In another experiment, spermatogenia were killed by 300 rads of x-rays and PA secretion was analyzed during the absence of desired germ cell classes. The results support the idea that upon their detachment from the basal lamina, preleptotene-stage primary spermatocytes have the major stimulatory action on the PA secretion of the seminiferous tubules. Other phenomena such as spermiation, phagocytosis of residual bodies or opening up of the Sertoli cell junctions seem to influence PA secretion to a lesser extent.

Animals↗

PubMed2001

Prevalence and significance of hepatitis B virus (HBV) pre-S mutants in serum and liver at different replicative stages of chronic HBV infection.

Several types of naturally occurring pre-S mutants in sera or liver tissues in patients with chronic hepatitis B virus (HBV) infection have been identified. To clarify the prevalence and significance of emergence of pre-S mutants, 140 sera and 18 resected livers from patients with HBV were studied. Replicative status was designated as high, intermediate, and low based on the HBV-DNA levels in serum or the expression of HBV antigens in liver. In vitro transfection and Western blot analysis were performed to characterize expression and secretion of HBsAg by the mutant constructs. Five major types (I to V) of pre-S deletion mutants in serum and liver and 2 types (VI and VII) in liver were identified. Pre-S mutant was 6.4% at high replicative phase, 13% at intermediate, and 37.5% at low or nonreplicative phases in serum. In livers, the same tendency existed: pre-S2 deletion mutants emerged and prevailed at a low replicative phase in hepatocytes that expressed a novel marginal pattern of HBsAg and usually clustered in groups. The deletion sequence of pre-S2 region coincides with human leukocyte antigen-restricted T- and B-cell epitopes. In vitro HBsAg was retained in the hepatocytes and synthesis and secretion of major surface antigen decreased for most of the pre-S mutants. Pre-S mutants prevailed with evolution of chronic HBV, probably under immune pressure. Emergence of pre-S mutants may account for the life-long persistence and discrepancy of HBsAg in serum and liver in HBV and may confer growth advantage in view of the clustering proliferation of hepatocytes harboring pre-S2 mutant.

Blotting, Western↗

PubMed1989

Cytoplasmic, nuclear, membrane-bound and secreted [35S]methionine-labelled polypeptide pattern in differentiating fibroblast stem cells in vitro.

Methods for the selective enrichment of various subpopulations of the human skin fibroblast cell line HH-8 have been developed. These methods permit the selection of homogeneous populations of the three mitotic fibroblast cell types MF I, II and III, and the four postmitotic cell types PMF IV, V, VI and VII. These seven cell types exhibit differentiation-dependent and cell-type-specific patterns of [35S]methionine-labelled polypeptides in total soluble cytoplasmic and nuclear proteins, also in membrane-bound proteins, and in secreted proteins. In the differentiation sequence MF II-MF III-PMF IV - PMF V - PMF VI 14 cell-type-specific marker proteins have been found in the cytoplasmic and nuclear fraction, also 24 cell-type-specific marker proteins have been found in the membrane-bound protein fraction, and 11 cell-type-specific marker proteins in the secreted protein fraction. Markers in spontaneously arising and experimentally selected or induced populations of a single fibroblast cell type were found to be identical.

Cell Differentiation↗

PubMed1996

The fibrinoids of the human placenta: origin, composition and functional relevance.

Placental fibrinoids are extracellularly deposited materials which are histologically glossy and acid staining, and can be found in every normal and pathological placenta at all stages of pregnancy. The amount of fibrinoid is, in general, independent of pregnancy outcome and fetal wellbeing. According to new findings, the classical histological term "fibrinoid" covers two distinctive extracellular matrices which differ as regards structure, composition and function. Fibrin-type fibrinoid is mostly composed of fibrin together with other molecules derived from blood clotting or degenerative processes. It is mainly a maternal blood-clot product which is used (a) to adapt the intervillous space to optimized flow conditions and (b) to control growth of the villous trees by encasing new villous branches which caused intervillous stasis or turbulence of maternal blood. Moreover, fibrin-type fibrinoid replaces degenerative syncytiotrophoblast at the maternofetal exchange surfaces, thus acting as a kind of substitute barrier. Matrix-type fibrinoid is a secretory product of invasive extravillous trophoblast cells. It shares some similarities with basement membranes, however, it is secreted in an apolar fashion, embedding the secreting cells. Like basement membranes, it contains laminins, collagen IV, and heparan sulfate. In addition, oncofetal fibronectins, vitronectin, and i-glycosylated molecules but no collagens I, III, and VII can be found. Matrix-type fibrinoid is thought to regulate trophoblast invasion by specific interactions with cell surface integrins. As a kind of "glue", it anchors the placenta to the uterine wall and seems to play an important role in materno-fetal immune interactions at this particular site. Both types of fibrinoid are usually co-localized, thus indicating close morphogenetic and functional interrelations.

Animals↗

PubMed1998

Coagulation factor VII Gln100 --> Arg. Amino acid substitution at the epidermal growth factor 2-protease domain interface results in severely reduced tissue factor binding and procoagulant function.

We have used recombinant mammalian expression and purification of the factor VII (FVII) variant Gln100 --> Arg (Q100RFVII) to study FVII deficiency in subjects with this mutation. Q100RFVII was secreted poorly in comparison with wild-type FVII (WTFVII) in a stable mammalian expression system, and purified variant protein was found to have undetectable clotting activity. Following activation by immobilized factor Xa, Q100RFVIIa had amidolytic activity similar to WTFVIIa in the absence of soluble tissue factor (sTF); however, unlike WTFVIIa no typical increase in activity was seen after addition of sTF. In a factor X activation assay using relipidated transmembrane truncated tissue factor (residues 1-243), Q100RFVIIa showed less than 5% of the ability of WTFVIIa to activate factor X. We performed direct binding analysis of WT and Q100RFVII/FVIIa to immobilized sTF using surface plasmon resonance, and severely reduced binding of both non-activated and activated Q100RFVII to sTF was seen, indicating a pronounced defect in tissue factor (TF) interaction with this variant. In the sTF-FVIIa crystal structure the candidate residue Gln100 is not in contact with TF but is at the epidermal growth factor 2-protease domain interface. We suggest that the mutation results in a global fold change severely reducing tissue factor interaction; mutation of FVII residues not directly involved in the interaction with TF may still result in variant FVII unable to take part in the initiation of coagulation.

Amino Acid Substitution↗