Role of the Degree of Oligomerization in the Structure and Function of Human Surfactant Protein A
The role of the degree of oligomerization in the structure and function of human surfactant protein A (SP-A) was investigated using a human SP-A1 mutant (SP-A1 ÎAVC,C6S ), expressed in mammalian cells, resulting from site-directed substitution of serine for Cys 6 and substitution of a functional si...
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| Veröffentlicht in: | The Journal of biological chemistry 2005-03, Vol.280 (9), p.7659-7670 |
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| creator | Fernando Sánchez-Barbero Jochen Strassner Rafael GarcÃa-Cañero Wolfram Steinhilber Cristina Casals |
| description | The role of the degree of oligomerization in the structure and function of human surfactant protein A (SP-A) was investigated
using a human SP-A1 mutant (SP-A1 ÎAVC,C6S ), expressed in mammalian cells, resulting from site-directed substitution of serine for Cys 6 and substitution of a functional signal peptide for the cysteine-containing SP-A signal sequence. This Cys 6 mutant lacked the NH 2 -terminal Ala â3 -Val â2 -Cys â1 (ÎAVC) extension present in some SP-A1 isoforms. SP-A1 ÎAVC,C6S was assembled exclusively as trimers as detected by electron microscopy and size exclusion chromatography. Trimeric SP-A1 ÎAVC,C6S was compared with supratrimeric SP-A1, which is structurally and functionally comparable to the octadecameric protein isolated
from human lung lavages. SP-A1 ÎAVC,C6S showed reduced thermal stability of the collagen domain, studied by circular dichroism, and increased susceptibility to trypsin
degradation. The T m was 32.7 °C for SP-A1 ÎAVC,C6S and 44.5 °C for SP-A1. Although SP-A1 ÎAVC,C6S was capable of binding to calcium, rough lipopolysaccharide, and phospholipid vesicles, this mutant was unable to induce
rough lipopolysaccharide and phospholipid vesicle aggregation, to enhance the interfacial adsorption of SP-B/SP-C-surfactant
membranes, and to undergo self-association in the presence of Ca 2+ . On the other hand, the lack of supratrimeric assembly hardly affected the ability of SP-A1 ÎAVC,C6S to inhibit the production of tumor necrosis factor-α by macrophage-like U937 cells stimulated with either smooth or rough
lipopolysaccharide. We conclude that supratrimeric assembly of human SP-A is essential for collagen triple helix stability
at physiological temperatures, protection against proteases, protein self-association, and SP-A-induced ligand aggregation.
The supratrimeric assembly is not essential for the binding of SP-A to ligands and anti-inflammatory effects of SP-A. |
| doi_str_mv | 10.1074/jbc.M410266200 |
| format | Article |
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using a human SP-A1 mutant (SP-A1 ÎAVC,C6S ), expressed in mammalian cells, resulting from site-directed substitution of serine for Cys 6 and substitution of a functional signal peptide for the cysteine-containing SP-A signal sequence. This Cys 6 mutant lacked the NH 2 -terminal Ala â3 -Val â2 -Cys â1 (ÎAVC) extension present in some SP-A1 isoforms. SP-A1 ÎAVC,C6S was assembled exclusively as trimers as detected by electron microscopy and size exclusion chromatography. Trimeric SP-A1 ÎAVC,C6S was compared with supratrimeric SP-A1, which is structurally and functionally comparable to the octadecameric protein isolated
from human lung lavages. SP-A1 ÎAVC,C6S showed reduced thermal stability of the collagen domain, studied by circular dichroism, and increased susceptibility to trypsin
degradation. The T m was 32.7 °C for SP-A1 ÎAVC,C6S and 44.5 °C for SP-A1. Although SP-A1 ÎAVC,C6S was capable of binding to calcium, rough lipopolysaccharide, and phospholipid vesicles, this mutant was unable to induce
rough lipopolysaccharide and phospholipid vesicle aggregation, to enhance the interfacial adsorption of SP-B/SP-C-surfactant
membranes, and to undergo self-association in the presence of Ca 2+ . On the other hand, the lack of supratrimeric assembly hardly affected the ability of SP-A1 ÎAVC,C6S to inhibit the production of tumor necrosis factor-α by macrophage-like U937 cells stimulated with either smooth or rough
lipopolysaccharide. We conclude that supratrimeric assembly of human SP-A is essential for collagen triple helix stability
at physiological temperatures, protection against proteases, protein self-association, and SP-A-induced ligand aggregation.
The supratrimeric assembly is not essential for the binding of SP-A to ligands and anti-inflammatory effects of SP-A.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1074/jbc.M410266200</identifier><identifier>PMID: 15615713</identifier><language>eng</language><publisher>United States: American Society for Biochemistry and Molecular Biology</publisher><subject>Adsorption ; Animals ; Anti-Inflammatory Agents - pharmacology ; Bronchoalveolar Lavage ; Calcium - chemistry ; Cell Membrane - metabolism ; CHO Cells ; Circular Dichroism ; Cloning, Molecular ; Cricetinae ; Cysteine - chemistry ; Dimerization ; Dose-Response Relationship, Drug ; Fluorescence Resonance Energy Transfer ; Humans ; Inflammation ; Kinetics ; Ligands ; Lipopolysaccharides - chemistry ; Macrophages - metabolism ; Microscopy, Electron ; Mutagenesis, Site-Directed ; Mutation ; Phospholipids - chemistry ; Protein Binding ; Protein Isoforms ; Protein Structure, Tertiary ; Pulmonary Surfactant-Associated Protein A - chemistry ; Pulmonary Surfactant-Associated Protein A - genetics ; Pulmonary Surfactant-Associated Protein A - physiology ; Recombinant Proteins - chemistry ; Spectrometry, Fluorescence ; Structure-Activity Relationship ; Temperature ; Time Factors ; Trypsin - pharmacology ; U937 Cells</subject><ispartof>The Journal of biological chemistry, 2005-03, Vol.280 (9), p.7659-7670</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c457t-56bd67f824b9e3da1fbcf2d0445478a19a7b8e2c2c98d37e4d12faefd4514ed73</citedby><cites>FETCH-LOGICAL-c457t-56bd67f824b9e3da1fbcf2d0445478a19a7b8e2c2c98d37e4d12faefd4514ed73</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/15615713$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Fernando Sánchez-Barbero</creatorcontrib><creatorcontrib>Jochen Strassner</creatorcontrib><creatorcontrib>Rafael GarcÃa-Cañero</creatorcontrib><creatorcontrib>Wolfram Steinhilber</creatorcontrib><creatorcontrib>Cristina Casals</creatorcontrib><title>Role of the Degree of Oligomerization in the Structure and Function of Human Surfactant Protein A</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>The role of the degree of oligomerization in the structure and function of human surfactant protein A (SP-A) was investigated
using a human SP-A1 mutant (SP-A1 ÎAVC,C6S ), expressed in mammalian cells, resulting from site-directed substitution of serine for Cys 6 and substitution of a functional signal peptide for the cysteine-containing SP-A signal sequence. This Cys 6 mutant lacked the NH 2 -terminal Ala â3 -Val â2 -Cys â1 (ÎAVC) extension present in some SP-A1 isoforms. SP-A1 ÎAVC,C6S was assembled exclusively as trimers as detected by electron microscopy and size exclusion chromatography. Trimeric SP-A1 ÎAVC,C6S was compared with supratrimeric SP-A1, which is structurally and functionally comparable to the octadecameric protein isolated
from human lung lavages. SP-A1 ÎAVC,C6S showed reduced thermal stability of the collagen domain, studied by circular dichroism, and increased susceptibility to trypsin
degradation. The T m was 32.7 °C for SP-A1 ÎAVC,C6S and 44.5 °C for SP-A1. Although SP-A1 ÎAVC,C6S was capable of binding to calcium, rough lipopolysaccharide, and phospholipid vesicles, this mutant was unable to induce
rough lipopolysaccharide and phospholipid vesicle aggregation, to enhance the interfacial adsorption of SP-B/SP-C-surfactant
membranes, and to undergo self-association in the presence of Ca 2+ . On the other hand, the lack of supratrimeric assembly hardly affected the ability of SP-A1 ÎAVC,C6S to inhibit the production of tumor necrosis factor-α by macrophage-like U937 cells stimulated with either smooth or rough
lipopolysaccharide. We conclude that supratrimeric assembly of human SP-A is essential for collagen triple helix stability
at physiological temperatures, protection against proteases, protein self-association, and SP-A-induced ligand aggregation.
The supratrimeric assembly is not essential for the binding of SP-A to ligands and anti-inflammatory effects of SP-A.</description><subject>Adsorption</subject><subject>Animals</subject><subject>Anti-Inflammatory Agents - pharmacology</subject><subject>Bronchoalveolar Lavage</subject><subject>Calcium - chemistry</subject><subject>Cell Membrane - metabolism</subject><subject>CHO Cells</subject><subject>Circular Dichroism</subject><subject>Cloning, Molecular</subject><subject>Cricetinae</subject><subject>Cysteine - chemistry</subject><subject>Dimerization</subject><subject>Dose-Response Relationship, Drug</subject><subject>Fluorescence Resonance Energy Transfer</subject><subject>Humans</subject><subject>Inflammation</subject><subject>Kinetics</subject><subject>Ligands</subject><subject>Lipopolysaccharides - chemistry</subject><subject>Macrophages - metabolism</subject><subject>Microscopy, Electron</subject><subject>Mutagenesis, Site-Directed</subject><subject>Mutation</subject><subject>Phospholipids - chemistry</subject><subject>Protein Binding</subject><subject>Protein Isoforms</subject><subject>Protein Structure, Tertiary</subject><subject>Pulmonary Surfactant-Associated Protein A - chemistry</subject><subject>Pulmonary Surfactant-Associated Protein A - genetics</subject><subject>Pulmonary Surfactant-Associated Protein A - physiology</subject><subject>Recombinant Proteins - chemistry</subject><subject>Spectrometry, Fluorescence</subject><subject>Structure-Activity Relationship</subject><subject>Temperature</subject><subject>Time Factors</subject><subject>Trypsin - pharmacology</subject><subject>U937 Cells</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2005</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpFkEFP3DAQRq2qqGxprz2iqAdu2XocO3aOCNhSiWoRFImb5djjXaMkBsdRBb--6e5KzGU0mvd9h0fIN6BLoJL_eGrt8jcHyuqaUfqBLICqqqwEPH4kC0oZlA0T6ph8HscnOg9v4BM5BlGDkFAtiLmLHRbRF3mLxSVuEu6udRc2sccU3kwOcSjCsAPuc5psnhIWZnDFahrs7jsHrqfeDMX9lLyx2Qy5uE0x4xw7_0KOvOlG_HrYJ-RhdfXn4rq8Wf_8dXF-U1ouZC5F3bpaesV422DlDPjWeuYo54JLZaAxslXILLONcpVE7oB5g95xARydrE7I2b73OcWXCces-zBa7DozYJxGDVIBCMVmcLkHbYrjmNDr5xR6k141UP1fqp6l6nepc-D00Dy1Pbp3_GBxBr7vgW3YbP-GhLoN0W6x10xR3WhZi6b6B7cAfqg</recordid><startdate>20050304</startdate><enddate>20050304</enddate><creator>Fernando Sánchez-Barbero</creator><creator>Jochen Strassner</creator><creator>Rafael GarcÃa-Cañero</creator><creator>Wolfram Steinhilber</creator><creator>Cristina Casals</creator><general>American Society for Biochemistry and Molecular Biology</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QP</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>RC3</scope></search><sort><creationdate>20050304</creationdate><title>Role of the Degree of Oligomerization in the Structure and Function of Human Surfactant Protein A</title><author>Fernando Sánchez-Barbero ; Jochen Strassner ; Rafael GarcÃa-Cañero ; Wolfram Steinhilber ; Cristina Casals</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c457t-56bd67f824b9e3da1fbcf2d0445478a19a7b8e2c2c98d37e4d12faefd4514ed73</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2005</creationdate><topic>Adsorption</topic><topic>Animals</topic><topic>Anti-Inflammatory Agents - pharmacology</topic><topic>Bronchoalveolar Lavage</topic><topic>Calcium - chemistry</topic><topic>Cell Membrane - metabolism</topic><topic>CHO Cells</topic><topic>Circular Dichroism</topic><topic>Cloning, Molecular</topic><topic>Cricetinae</topic><topic>Cysteine - chemistry</topic><topic>Dimerization</topic><topic>Dose-Response Relationship, Drug</topic><topic>Fluorescence Resonance Energy Transfer</topic><topic>Humans</topic><topic>Inflammation</topic><topic>Kinetics</topic><topic>Ligands</topic><topic>Lipopolysaccharides - chemistry</topic><topic>Macrophages - metabolism</topic><topic>Microscopy, Electron</topic><topic>Mutagenesis, Site-Directed</topic><topic>Mutation</topic><topic>Phospholipids - chemistry</topic><topic>Protein Binding</topic><topic>Protein Isoforms</topic><topic>Protein Structure, Tertiary</topic><topic>Pulmonary Surfactant-Associated Protein A - chemistry</topic><topic>Pulmonary Surfactant-Associated Protein A - genetics</topic><topic>Pulmonary Surfactant-Associated Protein A - physiology</topic><topic>Recombinant Proteins - chemistry</topic><topic>Spectrometry, Fluorescence</topic><topic>Structure-Activity Relationship</topic><topic>Temperature</topic><topic>Time Factors</topic><topic>Trypsin - pharmacology</topic><topic>U937 Cells</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Fernando Sánchez-Barbero</creatorcontrib><creatorcontrib>Jochen Strassner</creatorcontrib><creatorcontrib>Rafael GarcÃa-Cañero</creatorcontrib><creatorcontrib>Wolfram Steinhilber</creatorcontrib><creatorcontrib>Cristina Casals</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Fernando Sánchez-Barbero</au><au>Jochen Strassner</au><au>Rafael GarcÃa-Cañero</au><au>Wolfram Steinhilber</au><au>Cristina Casals</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Role of the Degree of Oligomerization in the Structure and Function of Human Surfactant Protein A</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>2005-03-04</date><risdate>2005</risdate><volume>280</volume><issue>9</issue><spage>7659</spage><epage>7670</epage><pages>7659-7670</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><abstract>The role of the degree of oligomerization in the structure and function of human surfactant protein A (SP-A) was investigated
using a human SP-A1 mutant (SP-A1 ÎAVC,C6S ), expressed in mammalian cells, resulting from site-directed substitution of serine for Cys 6 and substitution of a functional signal peptide for the cysteine-containing SP-A signal sequence. This Cys 6 mutant lacked the NH 2 -terminal Ala â3 -Val â2 -Cys â1 (ÎAVC) extension present in some SP-A1 isoforms. SP-A1 ÎAVC,C6S was assembled exclusively as trimers as detected by electron microscopy and size exclusion chromatography. Trimeric SP-A1 ÎAVC,C6S was compared with supratrimeric SP-A1, which is structurally and functionally comparable to the octadecameric protein isolated
from human lung lavages. SP-A1 ÎAVC,C6S showed reduced thermal stability of the collagen domain, studied by circular dichroism, and increased susceptibility to trypsin
degradation. The T m was 32.7 °C for SP-A1 ÎAVC,C6S and 44.5 °C for SP-A1. Although SP-A1 ÎAVC,C6S was capable of binding to calcium, rough lipopolysaccharide, and phospholipid vesicles, this mutant was unable to induce
rough lipopolysaccharide and phospholipid vesicle aggregation, to enhance the interfacial adsorption of SP-B/SP-C-surfactant
membranes, and to undergo self-association in the presence of Ca 2+ . On the other hand, the lack of supratrimeric assembly hardly affected the ability of SP-A1 ÎAVC,C6S to inhibit the production of tumor necrosis factor-α by macrophage-like U937 cells stimulated with either smooth or rough
lipopolysaccharide. We conclude that supratrimeric assembly of human SP-A is essential for collagen triple helix stability
at physiological temperatures, protection against proteases, protein self-association, and SP-A-induced ligand aggregation.
The supratrimeric assembly is not essential for the binding of SP-A to ligands and anti-inflammatory effects of SP-A.</abstract><cop>United States</cop><pub>American Society for Biochemistry and Molecular Biology</pub><pmid>15615713</pmid><doi>10.1074/jbc.M410266200</doi><tpages>12</tpages><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; EZB-FREE-00999 freely available EZB journals; PubMed Central; Alma/SFX Local Collection |
| subjects | Adsorption Animals Anti-Inflammatory Agents - pharmacology Bronchoalveolar Lavage Calcium - chemistry Cell Membrane - metabolism CHO Cells Circular Dichroism Cloning, Molecular Cricetinae Cysteine - chemistry Dimerization Dose-Response Relationship, Drug Fluorescence Resonance Energy Transfer Humans Inflammation Kinetics Ligands Lipopolysaccharides - chemistry Macrophages - metabolism Microscopy, Electron Mutagenesis, Site-Directed Mutation Phospholipids - chemistry Protein Binding Protein Isoforms Protein Structure, Tertiary Pulmonary Surfactant-Associated Protein A - chemistry Pulmonary Surfactant-Associated Protein A - genetics Pulmonary Surfactant-Associated Protein A - physiology Recombinant Proteins - chemistry Spectrometry, Fluorescence Structure-Activity Relationship Temperature Time Factors Trypsin - pharmacology U937 Cells |
| title | Role of the Degree of Oligomerization in the Structure and Function of Human Surfactant Protein A |
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