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Heterogeneity of recombinant human antithrombin III expressed in baby hamster kidney cells. Effect of glycosylation differences on heparin binding and structure
To determine the effects of differences in glycosylation on the structure and functional properties of recombinant human antithrombin (rHAT), we have characterized the properties of the recombinant protein overexpressed by baby hamster kidney cells. Three forms of rHAT, I-III, were isolated which di...
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| Published in: | The Journal of biological chemistry 1993-08, Vol.268 (23), p.17588-17596 |
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| Main Authors: | , , , , , |
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| Language: | English |
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| container_end_page | 17596 |
| container_issue | 23 |
| container_start_page | 17588 |
| container_title | The Journal of biological chemistry |
| container_volume | 268 |
| creator | Fan, B Crews, B C Turko, I V Choay, J Zettlmeissl, G Gettins, P |
| description | To determine the effects of differences in glycosylation on the structure and functional properties of recombinant human antithrombin
(rHAT), we have characterized the properties of the recombinant protein overexpressed by baby hamster kidney cells. Three
forms of rHAT, I-III, were isolated which differed in affinity for heparin. Form I had the lowest affinity and contained a
high proportion of highly branched complex carbohydrate. Form II had higher affinity and contained both complex and high mannose-type
chains. Form III had the highest affinity and was similar to form II in the type of carbohydrate present, but had a lower
level of glycosylation, consistent with the absence of carbohydrate at one of the four glycosylation sites. 1H NMR spectra
of plasma HAT and rHAT forms I-III suggested very similar protein structures for all forms. Heparin pentasaccharide produced
almost identical NMR perturbation difference spectra. The only functional difference found was in the rates of inactivation
of factor Xa. Forms II and III gave second order rate constants similar to that of plasma HAT, whereas form I gave a biphasic
inhibition, with the first phase having a rate about four times that of the other forms. We conclude that carbohydrate heterogeneity
does not alter the structure of the HAT polypeptide or the heparin-induced conformational change, but does affect the heparin
affinity and can alter the rate of proteinase inhibition. |
| doi_str_mv | 10.1016/s0021-9258(19)85373-5 |
| format | article |
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(rHAT), we have characterized the properties of the recombinant protein overexpressed by baby hamster kidney cells. Three
forms of rHAT, I-III, were isolated which differed in affinity for heparin. Form I had the lowest affinity and contained a
high proportion of highly branched complex carbohydrate. Form II had higher affinity and contained both complex and high mannose-type
chains. Form III had the highest affinity and was similar to form II in the type of carbohydrate present, but had a lower
level of glycosylation, consistent with the absence of carbohydrate at one of the four glycosylation sites. 1H NMR spectra
of plasma HAT and rHAT forms I-III suggested very similar protein structures for all forms. Heparin pentasaccharide produced
almost identical NMR perturbation difference spectra. The only functional difference found was in the rates of inactivation
of factor Xa. Forms II and III gave second order rate constants similar to that of plasma HAT, whereas form I gave a biphasic
inhibition, with the first phase having a rate about four times that of the other forms. We conclude that carbohydrate heterogeneity
does not alter the structure of the HAT polypeptide or the heparin-induced conformational change, but does affect the heparin
affinity and can alter the rate of proteinase inhibition.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1016/s0021-9258(19)85373-5</identifier><identifier>PMID: 8349638</identifier><language>eng</language><publisher>United States: American Society for Biochemistry and Molecular Biology</publisher><subject>Animals ; Antithrombin III - chemistry ; Antithrombin III - metabolism ; Carbohydrates - analysis ; Cell Line ; Cricetinae ; Electrophoresis, Polyacrylamide Gel ; Factor Xa Inhibitors ; Glycosylation ; Heparin - chemistry ; Heparin - metabolism ; Humans ; Kinetics ; Magnetic Resonance Spectroscopy ; Protein Binding ; Recombinant Proteins - chemistry ; Recombinant Proteins - metabolism</subject><ispartof>The Journal of biological chemistry, 1993-08, Vol.268 (23), p.17588-17596</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c446t-5d44138308d5e6155fcd021ea4e75f823b4dcaf7cc8d0810030231a335fccd0f3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>315,783,787,27936,27937</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/8349638$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Fan, B</creatorcontrib><creatorcontrib>Crews, B C</creatorcontrib><creatorcontrib>Turko, I V</creatorcontrib><creatorcontrib>Choay, J</creatorcontrib><creatorcontrib>Zettlmeissl, G</creatorcontrib><creatorcontrib>Gettins, P</creatorcontrib><title>Heterogeneity of recombinant human antithrombin III expressed in baby hamster kidney cells. Effect of glycosylation differences on heparin binding and structure</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>To determine the effects of differences in glycosylation on the structure and functional properties of recombinant human antithrombin
(rHAT), we have characterized the properties of the recombinant protein overexpressed by baby hamster kidney cells. Three
forms of rHAT, I-III, were isolated which differed in affinity for heparin. Form I had the lowest affinity and contained a
high proportion of highly branched complex carbohydrate. Form II had higher affinity and contained both complex and high mannose-type
chains. Form III had the highest affinity and was similar to form II in the type of carbohydrate present, but had a lower
level of glycosylation, consistent with the absence of carbohydrate at one of the four glycosylation sites. 1H NMR spectra
of plasma HAT and rHAT forms I-III suggested very similar protein structures for all forms. Heparin pentasaccharide produced
almost identical NMR perturbation difference spectra. The only functional difference found was in the rates of inactivation
of factor Xa. Forms II and III gave second order rate constants similar to that of plasma HAT, whereas form I gave a biphasic
inhibition, with the first phase having a rate about four times that of the other forms. We conclude that carbohydrate heterogeneity
does not alter the structure of the HAT polypeptide or the heparin-induced conformational change, but does affect the heparin
affinity and can alter the rate of proteinase inhibition.</description><subject>Animals</subject><subject>Antithrombin III - chemistry</subject><subject>Antithrombin III - metabolism</subject><subject>Carbohydrates - analysis</subject><subject>Cell Line</subject><subject>Cricetinae</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Factor Xa Inhibitors</subject><subject>Glycosylation</subject><subject>Heparin - chemistry</subject><subject>Heparin - metabolism</subject><subject>Humans</subject><subject>Kinetics</subject><subject>Magnetic Resonance Spectroscopy</subject><subject>Protein Binding</subject><subject>Recombinant Proteins - chemistry</subject><subject>Recombinant Proteins - metabolism</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1993</creationdate><recordtype>article</recordtype><recordid>eNo9UcFu1DAQtRCoLIVPqOQDQnBIsWM7cY6oKnSlShwAiZvl2JONIbEX2xHN3_CpON1VfbFn3ps3nnkIXVFyTQltPiZCalp1tZDvafdBCtaySjxDO0okq5igP5-j3RPlJXqV0i9SDu_oBbqQjHcNkzv07w4yxHAADy6vOAw4gglz77z2GY_LrD0uL5fH-JjF-_0ew8MxQkpgcUn0ul_xqOdUdPBvZz2s2MA0pWt8Owxg8iZ6mFYT0jrp7ILH1hUggjeQcAlHOOq4KTlvnT-UfhanHBeTlwiv0YtBTwnenO9L9OPz7febu-r-65f9zaf7ynDe5EpYzimTjEgroKFCDMaW4UFzaMUga9Zza_TQGiMtkZQQRmpGNWOFWJgDu0TvTrrHGP4skLKaXdrm0B7CklQrpORNWxeiOBFNDClFGNQxulnHVVGiNmfUt23talu7op16dEaJUnd1brD0M9inqrMVBX97wkd3GP-6CKp3wYwwq7qRqmaKbl9g_wHj05l5</recordid><startdate>19930815</startdate><enddate>19930815</enddate><creator>Fan, B</creator><creator>Crews, B C</creator><creator>Turko, I V</creator><creator>Choay, J</creator><creator>Zettlmeissl, G</creator><creator>Gettins, P</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>7X8</scope></search><sort><creationdate>19930815</creationdate><title>Heterogeneity of recombinant human antithrombin III expressed in baby hamster kidney cells. Effect of glycosylation differences on heparin binding and structure</title><author>Fan, B ; Crews, B C ; Turko, I V ; Choay, J ; Zettlmeissl, G ; Gettins, P</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c446t-5d44138308d5e6155fcd021ea4e75f823b4dcaf7cc8d0810030231a335fccd0f3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1993</creationdate><topic>Animals</topic><topic>Antithrombin III - chemistry</topic><topic>Antithrombin III - metabolism</topic><topic>Carbohydrates - analysis</topic><topic>Cell Line</topic><topic>Cricetinae</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Factor Xa Inhibitors</topic><topic>Glycosylation</topic><topic>Heparin - chemistry</topic><topic>Heparin - metabolism</topic><topic>Humans</topic><topic>Kinetics</topic><topic>Magnetic Resonance Spectroscopy</topic><topic>Protein Binding</topic><topic>Recombinant Proteins - chemistry</topic><topic>Recombinant Proteins - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Fan, B</creatorcontrib><creatorcontrib>Crews, B C</creatorcontrib><creatorcontrib>Turko, I V</creatorcontrib><creatorcontrib>Choay, J</creatorcontrib><creatorcontrib>Zettlmeissl, G</creatorcontrib><creatorcontrib>Gettins, P</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Fan, B</au><au>Crews, B C</au><au>Turko, I V</au><au>Choay, J</au><au>Zettlmeissl, G</au><au>Gettins, P</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Heterogeneity of recombinant human antithrombin III expressed in baby hamster kidney cells. Effect of glycosylation differences on heparin binding and structure</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>1993-08-15</date><risdate>1993</risdate><volume>268</volume><issue>23</issue><spage>17588</spage><epage>17596</epage><pages>17588-17596</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><abstract>To determine the effects of differences in glycosylation on the structure and functional properties of recombinant human antithrombin
(rHAT), we have characterized the properties of the recombinant protein overexpressed by baby hamster kidney cells. Three
forms of rHAT, I-III, were isolated which differed in affinity for heparin. Form I had the lowest affinity and contained a
high proportion of highly branched complex carbohydrate. Form II had higher affinity and contained both complex and high mannose-type
chains. Form III had the highest affinity and was similar to form II in the type of carbohydrate present, but had a lower
level of glycosylation, consistent with the absence of carbohydrate at one of the four glycosylation sites. 1H NMR spectra
of plasma HAT and rHAT forms I-III suggested very similar protein structures for all forms. Heparin pentasaccharide produced
almost identical NMR perturbation difference spectra. The only functional difference found was in the rates of inactivation
of factor Xa. Forms II and III gave second order rate constants similar to that of plasma HAT, whereas form I gave a biphasic
inhibition, with the first phase having a rate about four times that of the other forms. We conclude that carbohydrate heterogeneity
does not alter the structure of the HAT polypeptide or the heparin-induced conformational change, but does affect the heparin
affinity and can alter the rate of proteinase inhibition.</abstract><cop>United States</cop><pub>American Society for Biochemistry and Molecular Biology</pub><pmid>8349638</pmid><doi>10.1016/s0021-9258(19)85373-5</doi><tpages>9</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | The Journal of biological chemistry, 1993-08, Vol.268 (23), p.17588-17596 |
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| language | eng |
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| source | ScienceDirect |
| subjects | Animals Antithrombin III - chemistry Antithrombin III - metabolism Carbohydrates - analysis Cell Line Cricetinae Electrophoresis, Polyacrylamide Gel Factor Xa Inhibitors Glycosylation Heparin - chemistry Heparin - metabolism Humans Kinetics Magnetic Resonance Spectroscopy Protein Binding Recombinant Proteins - chemistry Recombinant Proteins - metabolism |
| title | Heterogeneity of recombinant human antithrombin III expressed in baby hamster kidney cells. Effect of glycosylation differences on heparin binding and structure |
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