Evidence for a two-step mechanism involved in the formation of covalent HC·TSG-6 complexes

Kristian W. Sanggaard; Carsten S. Sonne-Schmidt; Christian Jacobsen; Ida B. Thøgersen; Zuzana Valnickova; Hans Georg Wisniewski; Jan J. Enghild

doi:10.1021/bi060106s

Evidence for a two-step mechanism involved in the formation of covalent HC·TSG-6 complexes

Kristian W. Sanggaard, Carsten S. Sonne-Schmidt, Christian Jacobsen, Ida B. Thøgersen, Zuzana Valnickova, Hans Georg Wisniewski, Jan J. Enghild^*

^*Corresponding author af dette arbejde

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avis › Tidsskriftartikel › Forskning › peer review

27 Citationer (Scopus)

Abstract

IαI and TSG-6 interact to form a covalent bond between the C-terminal Asp α-carbon of an IαI heavy chain (HC) and an unknown component of TSG-6. This event disrupts the protein-glycosaminoglycan-protein (PGP) cross-link and dissociates IαI. In simple terms the interaction involves 5 components: (i) the IαI HCs, (ii) bikunin, (iii) chondroitin sulfate chain, (iv) TSG-6, and (v) divalent cations. To understand the molecular mechanism of complex formation, the effect of these were separately examined. The data show that although the mature covalent cross-link between the HCs and TSG-6 only involves the C-terminal Asp residue, the native fold of both IαI and TSG-6 was essential for the reaction to occur. Similarly, complex formation was prevented if the chondroitin sulfate chain was cleaved, releasing bikunin but maintaining the HC1 and HC2 PGP cross-links. In contrast, releasing the majority of the bikunin protein moiety by limited proteolysis did not prevent complex formation. An analysis of the divalent-cation requirements revealed two distinct interactions between IαI and TSG-6: (i) a noncovalent manganese, magnesium, or calcium-independent interaction between TSG-6 and the chondroitin sulfate chain (K_d 180 nM) and (ii) a covalent manganese, magnesium, or calcium-dependent interaction generating HC1·TSG-6, HC2·TSG-6, and high molecular weight (HMW) IαI. Significantly, both free TSG-6 and HC·TSG-6 complexes were able to bind the chondroitin sulfate chain suggesting that the sites on TSG-6 were distinct. On the basis of these findings, we propose a two-step reaction mechanism involving two putative binding sites. Initially, a cation-independent interaction between TSG-6 and the chondroitin sulfate chain is formed at site 1. Subsequently, a cation-dependent transesterification occurs, generating the covalent HC·TSG-6 cross-link at another site, site 2.

Originalsprog	Engelsk
Tidsskrift	Biochemistry
Vol/bind	45
Nummer	24
Sider (fra-til)	7661-7668
Antal sider	8
ISSN	0006-2960
DOI	https://doi.org/10.1021/bi060106s
Status	Udgivet - 20 jun. 2006

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title = "Evidence for a two-step mechanism involved in the formation of covalent HC·TSG-6 complexes",

abstract = "IαI and TSG-6 interact to form a covalent bond between the C-terminal Asp α-carbon of an IαI heavy chain (HC) and an unknown component of TSG-6. This event disrupts the protein-glycosaminoglycan-protein (PGP) cross-link and dissociates IαI. In simple terms the interaction involves 5 components: (i) the IαI HCs, (ii) bikunin, (iii) chondroitin sulfate chain, (iv) TSG-6, and (v) divalent cations. To understand the molecular mechanism of complex formation, the effect of these were separately examined. The data show that although the mature covalent cross-link between the HCs and TSG-6 only involves the C-terminal Asp residue, the native fold of both IαI and TSG-6 was essential for the reaction to occur. Similarly, complex formation was prevented if the chondroitin sulfate chain was cleaved, releasing bikunin but maintaining the HC1 and HC2 PGP cross-links. In contrast, releasing the majority of the bikunin protein moiety by limited proteolysis did not prevent complex formation. An analysis of the divalent-cation requirements revealed two distinct interactions between IαI and TSG-6: (i) a noncovalent manganese, magnesium, or calcium-independent interaction between TSG-6 and the chondroitin sulfate chain (Kd 180 nM) and (ii) a covalent manganese, magnesium, or calcium-dependent interaction generating HC1·TSG-6, HC2·TSG-6, and high molecular weight (HMW) IαI. Significantly, both free TSG-6 and HC·TSG-6 complexes were able to bind the chondroitin sulfate chain suggesting that the sites on TSG-6 were distinct. On the basis of these findings, we propose a two-step reaction mechanism involving two putative binding sites. Initially, a cation-independent interaction between TSG-6 and the chondroitin sulfate chain is formed at site 1. Subsequently, a cation-dependent transesterification occurs, generating the covalent HC·TSG-6 cross-link at another site, site 2.",

author = "Sanggaard, {Kristian W.} and Sonne-Schmidt, {Carsten S.} and Christian Jacobsen and Th{\o}gersen, {Ida B.} and Zuzana Valnickova and Wisniewski, {Hans Georg} and Enghild, {Jan J.}",

year = "2006",

month = jun,

day = "20",

doi = "10.1021/bi060106s",

language = "English",

volume = "45",

pages = "7661--7668",

journal = "Biochemistry",

issn = "0006-2960",

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T1 - Evidence for a two-step mechanism involved in the formation of covalent HC·TSG-6 complexes

AU - Sanggaard, Kristian W.

AU - Sonne-Schmidt, Carsten S.

AU - Jacobsen, Christian

AU - Thøgersen, Ida B.

AU - Valnickova, Zuzana

AU - Wisniewski, Hans Georg

AU - Enghild, Jan J.

PY - 2006/6/20

Y1 - 2006/6/20

N2 - IαI and TSG-6 interact to form a covalent bond between the C-terminal Asp α-carbon of an IαI heavy chain (HC) and an unknown component of TSG-6. This event disrupts the protein-glycosaminoglycan-protein (PGP) cross-link and dissociates IαI. In simple terms the interaction involves 5 components: (i) the IαI HCs, (ii) bikunin, (iii) chondroitin sulfate chain, (iv) TSG-6, and (v) divalent cations. To understand the molecular mechanism of complex formation, the effect of these were separately examined. The data show that although the mature covalent cross-link between the HCs and TSG-6 only involves the C-terminal Asp residue, the native fold of both IαI and TSG-6 was essential for the reaction to occur. Similarly, complex formation was prevented if the chondroitin sulfate chain was cleaved, releasing bikunin but maintaining the HC1 and HC2 PGP cross-links. In contrast, releasing the majority of the bikunin protein moiety by limited proteolysis did not prevent complex formation. An analysis of the divalent-cation requirements revealed two distinct interactions between IαI and TSG-6: (i) a noncovalent manganese, magnesium, or calcium-independent interaction between TSG-6 and the chondroitin sulfate chain (Kd 180 nM) and (ii) a covalent manganese, magnesium, or calcium-dependent interaction generating HC1·TSG-6, HC2·TSG-6, and high molecular weight (HMW) IαI. Significantly, both free TSG-6 and HC·TSG-6 complexes were able to bind the chondroitin sulfate chain suggesting that the sites on TSG-6 were distinct. On the basis of these findings, we propose a two-step reaction mechanism involving two putative binding sites. Initially, a cation-independent interaction between TSG-6 and the chondroitin sulfate chain is formed at site 1. Subsequently, a cation-dependent transesterification occurs, generating the covalent HC·TSG-6 cross-link at another site, site 2.

AB - IαI and TSG-6 interact to form a covalent bond between the C-terminal Asp α-carbon of an IαI heavy chain (HC) and an unknown component of TSG-6. This event disrupts the protein-glycosaminoglycan-protein (PGP) cross-link and dissociates IαI. In simple terms the interaction involves 5 components: (i) the IαI HCs, (ii) bikunin, (iii) chondroitin sulfate chain, (iv) TSG-6, and (v) divalent cations. To understand the molecular mechanism of complex formation, the effect of these were separately examined. The data show that although the mature covalent cross-link between the HCs and TSG-6 only involves the C-terminal Asp residue, the native fold of both IαI and TSG-6 was essential for the reaction to occur. Similarly, complex formation was prevented if the chondroitin sulfate chain was cleaved, releasing bikunin but maintaining the HC1 and HC2 PGP cross-links. In contrast, releasing the majority of the bikunin protein moiety by limited proteolysis did not prevent complex formation. An analysis of the divalent-cation requirements revealed two distinct interactions between IαI and TSG-6: (i) a noncovalent manganese, magnesium, or calcium-independent interaction between TSG-6 and the chondroitin sulfate chain (Kd 180 nM) and (ii) a covalent manganese, magnesium, or calcium-dependent interaction generating HC1·TSG-6, HC2·TSG-6, and high molecular weight (HMW) IαI. Significantly, both free TSG-6 and HC·TSG-6 complexes were able to bind the chondroitin sulfate chain suggesting that the sites on TSG-6 were distinct. On the basis of these findings, we propose a two-step reaction mechanism involving two putative binding sites. Initially, a cation-independent interaction between TSG-6 and the chondroitin sulfate chain is formed at site 1. Subsequently, a cation-dependent transesterification occurs, generating the covalent HC·TSG-6 cross-link at another site, site 2.

UR - http://www.scopus.com/inward/record.url?scp=33745173107&partnerID=8YFLogxK

U2 - 10.1021/bi060106s

DO - 10.1021/bi060106s

M3 - Journal article

C2 - 16768462

AN - SCOPUS:33745173107

SN - 0006-2960

VL - 45

SP - 7661

EP - 7668

JO - Biochemistry

JF - Biochemistry

IS - 24

ER -

Evidence for a two-step mechanism involved in the formation of covalent HC·TSG-6 complexes

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