Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.3 (collagenase)
 18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

As the most abundant glycoprotein component of pulmonary surfactant, SP-A (Mr = 30,000-36,000) plays a central role in the organization of phospholipid bilayers in the alveolar air space. SP-A, isolated from lung lavage, exists in oligomeric forms (N = 6, 12, 18, ...), mediated by collagen-like triple helices and intermolecular disulfide bonds. These protein-protein interactions, involving the amino-terminal domain of SP-A, are hypothesized to facilitate the alignment of surfactant lipid bilayers into unique tubular myelin structures. SP-A reorganization of surfactant lipid was assessed in vitro by quantitating the calcium-dependent light scattering properties of lipid vesicle suspensions induced by SP-A. Accelerated aggregation of unilamellar vesicles required SP-A and at least 3 mM free calcium. The initial rate of aggregation was proportional to the concentration of canine SP-A over lipid:protein molar ratios ranging from 200:1 to 5000:1. Digestion with bacterial collagenase or incubation with dithiothreitol (DTT) completely blocked lipid aggregation activity. Both treatments decreased the binding of SP-A to phospholipids. The conditions used in the DTT experiments (10 mM DTT, nondenaturing Tris buffer, 37 degrees C) resulted in the selective reduction and 14C-alkylation of the intermolecular disulfide bond involving residue 9Cys, whereas the four cysteines found in the noncollagenous domain of SP-A were inefficiently alkylated with [14C]-iodoacetate. HPLC analysis of tryptic SP-A peptides revealed that these four cysteine residues participate in intramolecular disulfide bond formation (138Cys-229Cys and 207Cys-221Cys). Our data demonstrate the importance of the quaternary structure (triple helix and intermolecular disulfide bond) of SP-A for the aggregation of unilamellar phospholipid vesicles.
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PMID:Intermolecular cross-links mediate aggregation of phospholipid vesicles by pulmonary surfactant protein SP-A. 198 71

SP-A, a glycoprotein of pulmonary surfactant, consists of an NH2-terminal domain containing a collagen-like sequence and a COOH-terminal domain with sequence homology to several Ca2(+)-dependent lectins. We have compared the size, thermal stability, and secondary structure of recombinant SP-A, the product of a fibroblast line transfected with a single human gene encoding SP-A, with natural SP-A isolated from canine and human lungs. Our results suggest both recombinant and natural SP-A are assembled as large oligomers. More variability in the degree of oligomerization was observed with recombinant human SP-A than with natural canine SP-A. As shown by collagenase digestion, the full assembly of protein subunits was dependent on an intact collagen-like domain. The cysteines in the noncollagen domain of SP-A form intrachain bonds between residues 135-226 and 204-218. The circular dichroism spectra of both recombinant and natural SP-A were consistent with the presence of a collagen-like triple helix. As determined by the change in ellipticity at 205 nm, the thermal transition temperatures of canine, natural human, and recombinant SP-A were 51.5, 52.3, and 42.0 degrees C, respectively. These results suggest differences in the assembly and stability of the natural and recombinant proteins.
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PMID:Studies of the structure of lung surfactant protein SP-A. 261 Feb 70

The results of a large number of studies indicate that pulmonary surfactant contains a unique protein whose principal isoform has a molecular weight of about 30,000, and whose presence in surfactant is associated with important metabolic and physicochemical properties. This protein, SP-A, as isolated from canine surfactant, contains a domain of 24 repeating triplets of Gly-X-Y, similar to that found in collagens. These studies were undertaken to determine whether SP-A forms a collagen-like triple helix when in solution, and to describe certain aspects of its size and shape. Our experiments were done on SP-A extracted by two different methods from canine surfactant, and on SP-A produced by molecular cloning. The results from all three preparations were similar. The circular dichroism of the complete protein was characterized by a relatively large negative ellipticity at 205 nm, with a negative shoulder ranging from 215 to 230 nm. There was no positive ellipticity, and the spectrum was not characteristic of collagen. Trypsin hydrolysis resulted in a fragment with peak negative ellipticity at about 200 nm, without the negative shoulder. Further hydrolysis of this fragment with pepsin resulted in a CD spectrum similar to that of collagen. The spectrum of the collagen-like fragment was reversibly sensitive to heating to 50 degrees C, and was irreversibly lost after treatment with bacterial collagenase. SP-A migrated on molecular sieving gels with an equivalent Stokes radius of 110 to 120 A, and had a sedimentation coefficient of 14 S. Using these data we calculate a molecular weight of about 700,000. The hydrodynamic characteristics can be approximated as a prolate ellipsoid of revolution having an axial ratio of about 20. We conclude that SP-A aggregates into a complex of 18 monomers, which may form six triple-helices. The shape of the complex is considerably more globular than collagen and is not consistent with end-to-end binding of the helices to form fibrous structures.
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PMID:Aspects of secondary and quaternary structure of surfactant protein A from canine lung. 291 54

Studies of the regulation of surfactant lipoprotein metabolism and secretion and surfactant protein gene expression have been hampered by the lack of a cell culture system in which the phenotypic properties of type II cells are maintained. We have developed a primary culture system that facilitates the maintenance of a number of morphologic and biochemical properties of type II pneumonocytes for up to 2 wk. Cells were isolated by collagenase digestion of midgestation human fetal lung tissue that had been maintained in organ culture in the presence of dibutyryl cyclic AMP (Bt2cAMP) for 5 days. The isolated cells were enriched for epithelial components by treatment with DEAE-dextran, plated on an extracellular matrix (ECM) derived from Madin-Darby canine kidney (MDCK) cells, and incubated at an air/liquid interface in a minimal amount of culture medium containing Bt2cAMP. The cell cultures were comprised of islands of round epithelial-like cells containing numerous dense osmiophilic granules, surrounded by sparse spindle-shaped cells with the appearance of fibroblasts. Ultrastructural examination revealed that the osmiophilic granules had the appearance of lamellar bodies, the distinguishing feature of type II pneumonocytes. Additionally, the cultures maintained elevated levels of SP-A gene expression for up to 2 wk. The expression of mRNAs encoding SP-A, SP-B, and SP-C were regulated in the cultured cells by glucocorticoids and cyclic AMP in a manner similar to that observed in fetal lung tissue in organ culture. The differentiated phenotype was most apparent when the cells were cultured at an air/liquid interface. In order to utilize the cultured type II cells for study of the effects of overexpression of various proteins and for promoter analysis, it is of essence to transfect DNA constructs into these cells with high efficiency. Unfortunately, we found the cells to be refractory to efficient transfer of DNA using conventional methods (i.e., lipofection, electroporation, or calcium phosphate-mediated transfection). However, replication-defective recombinant human adenoviruses were found to provide a highly efficient means of introducing DNA into the type II pneumonocytes. Furthermore, we observed in type II cell-enriched cultures infected with recombinant adenoviruses containing the lacZ gene under control of a cytomegalovirus promoter, that beta-galactosidase was expressed uniformly in the islands of type II cells and surrounding fibroblasts. By contrast, in cultures infected with recombinant adenoviruses containing the human growth hormone (hGH) gene under control of the SP-A gene promoter and 5'-flanking region, hGH was expressed only in the type II cells. Thus, this culture system provides an excellent means for identifying genomic elements that mediate type II cell-specific gene expression.
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PMID:Primary cell culture of human type II pneumonocytes: maintenance of a differentiated phenotype and transfection with recombinant adenoviruses. 940 54

This thesis is based on nine papers and a review on the collectins and collectin receptors in innate immunity. The collectins are a family of proteins in which the individual chains consist of a C-type lectin domain attached to a collagen domain via an alpha-coiled neck region. The chains are organized into a triple collagen helix and oligomerized through N-terminally located cysteines. The collectins have a dual function: one is to bind specifically to carbohydrate structures on the surface of a pathogen; the other is subsequently to recruit other cells and molecules to destroy the pathogen. The C-type lectin domains contain 110-130 amino-acid residues arranged in a conserved sequence pattern which allows the domain to fold into a well-defined tertiary structure. Five collectins have been described. Lung surfactant proteins A and D (SP-A and SP-D) are mainly found in the surfactant coating the luminal surface of the pulmonary epithelial cells, but are also produced by cells lining the gastrointestinal tract. Mannan-binding lectin (MBL), conglutinin and collectin-43 (CL-43) are serum proteins produced by the liver. Conglutinin and CL-43 have so far only been found in Bovidae. The collectins are involved in innate, nonadaptive immune defense. They bind to microbial surface carbohydrates, inducing aggregation and thereby impeding infectivity or mediating phagocytosis through specific receptors on the phagocytes. After binding microbial carbohydrate, MBL can activate the complement system through a newly discovered pathway which makes use of two serine proteases (MASP-1 and MASP-2) to activate the complement factors C4 and C2. In man, low serum MBL concentrations resulting from mutations in the collagen region are associated with a common opsonic defect. CL-43 was identified as a new collectin by its calcium-dependent binding to mannan and by its M(r) of 43 kDa in the reduced state on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The lectin was isolated by affinity chromatography on mannan-Sepharose, absorption with rabbit anti-bovine Ig coupled to Sepharose-4B and ion-exchange chromatography. CL-43 shows an apparent molecular mass of 120 kDa in the unreduced state on SDS-PAGE and elutes with an apparent molecular mass of 750 kDa on gel chromatography under nondissociating conditions. Amino-acid analysis and susceptibility to collagenase digestion indicated that CL-43 was a collectin. Electron microscopy of purified CL-43 revealed only rod-like monomer subunits 37.4 nm long. Two-dimensional gel electrophoresis showed that CL-43 has two isoforms of pI 4.9 and 5.3 respectively, corresponding to the native form of CL-43 and a truncated form which lacks the first 9 amino-acid residues. The N-terminal amino-acid sequence of CL-43 was used to design primers for PCR with a bovine liver cDNA as template. The cDNA of CL-43 was cloned and the open reading frame was found to encode a protein of 301 amino-acid residues, including an N-terminal region of 28 residues, a collagen region of 114 residues and a neck-CRD region of 159 residues. The amino-acid sequence of CL-43 shows 74% identity with bovine conglutinin and 70% identity with bovine SP-D, but the collagen region is considerably shorter than those of conglutinin and SP-D. Northern blot analysis showed that CL-43 was only synthesized in bovine liver, no signal being detected in a variety of other bovine tissues, including lung. No cross-hybridizing signals were detected in mRNA from ovine, human, rat or mouse liver. Since CL-43 and conglutinin have only been detected in members of the Bovidae, it is probable that an ancestral gene of these two proteins was first derived from a SP-D-like gene and that this ancestral gene underwent duplication during evolution. The carbohydrate binding profile of CL-43 was analyzed by an inhibition assay with biotinylated CL-43, using solid-phase mannan as the ligand. (ABSTRACT TRUNCATED)
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PMID:Collectins and collectin receptors in innate immunity. 1102 Dec 54

We report a simplified culture system for human fetal lung type II cells that maintains surfactant expression. Type II cells isolated from explant cultures of hormone-treated lungs (18-22 wk gestation) by collagenase + trypsin digestion were cultured on plastic for 4 days in serum-free medium containing dexamethasone (Dex, 10 nM) + 8-bromo-cAMP (0.1 mM + isobutylmethylxanthine (0.1 mM) or were untreated (control). Surfactant protein (SP) mRNAs decreased markedly in control cells between days 1 and 4 of culture, but mRNA levels were high in treated cells on day) 4 (SP-A, SP-B, SP-C, SP-D; 600%, 100%, 85%, 130% of day 0 content, respectively). Dex or cAMP alone increased SP-B, SP-C, and SP-D mRNAs and together had additive effects. The greatest increase in SP-A mRNA occurred with cAMP alone. Treated cells processed pro-SP-B and pro-SP-C proteins to mature forms and had a higher rate of phosphatidylcholine (PC) synthesis (2-fold) and higher saturation of PC (approximately 34% versus 27%) than controls. Only treated cells maintained secretagogue-responsive phospholipid synthesis. By electron microscopy, the treated cells retained lamellar bodies and extensive microvilli. We conclude that Dex and cAMP additively stimulate expression of surfactant components in isolated fetal type II cells, providing a simplified culture system for investigation of surfactant-related, and perhaps other, type II cell functions.
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PMID:Maintenance of differentiated function of the surfactant system in human fetal lung type II epithelial cells cultured on plastic. 1155 39

A culture system designed to maintain the differentiated characteristics of rat type II cells based on protocols used for human fetal lung pneumocytes was investigated. Type II cells were isolated either from adult rats with elastase (adult type II cells) or from young rats (4-11 days postnatal) with collagenase and trypsin (young type II cells) and were incubated with dexamethasone (Dex, 10 nM) and cAMP (0.1 mM). By day 4 of culture with hormone treatment, the mRNA levels in adult type II cells were less than 3% of day 0 values, whereas surfactant protein (SP)-A protein content was 26%. However, young type II cells maintained lamellar bodies and microvilli and secreted phospholipid in response to ATP. SP-A, -B, and -C mRNA levels were elevated to 159, 350, and 39%, respectively, of day 0 values with a synergistic response to Dex and cAMP, whereas SP-A protein content rose to 119%. Surfactant mRNA and protein did not recover in cells cultured without hormones. This cell culture system restored surfactant components in rat type II cells.
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PMID:Recovery of rat type II cell surfactant components during primary cell culture. 1179 31