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Recombinant human laminin-5 domains - Effects of heterotrimerization, proteolytic processing, and N-glycosylation on alpha(3)beta(1) integrin binding

Recombinant human laminin-5 domains - Effects of heterotrimerization, proteolytic processing, and N-glycosylation on alpha(3)beta(1) integrin binding
Recombinant human laminin-5 domains - Effects of heterotrimerization, proteolytic processing, and N-glycosylation on alpha(3)beta(1) integrin binding
Human laminin-5 fragments, comprising the heterotrimeric C-terminal part of the coiled-coil (CC) domain and the globular (G) domain with defined numbers of LG subdomains, were produced recombinantly. The 3' chain with all five LG subdomains was processed proteolytically in a manner similar to the wild-type 3 chain. Conditions were established under which the proteolytic cleavage was either inhibited in cell culture or was brought to completion in vitro. The shorter chains of the laminin-5CCG molecule, 3'and 2', produced in a bacterial expression system associated into heterodimers, which then combined spontaneously with the 3' chains in vitro to form heterotrimeric laminin-5CCG molecules. Only heterotrimeric laminin-5CCG with at least subdomains LG1–3, but not the single chains, supported binding of soluble 31 integrin, proving the coiled-coil domain of laminin-5 to be essential for its interaction with 31 integrin. The N-glycosylation sites in wild-type 3 chain were mapped by mass spectrometry. Their location in a structural model of the LG domain suggested that large regions on both faces of the LG1 and LG2 domains are inaccessible by other proteins. However, neither heterotrimerization nor 31 integrin binding was affected by the loss of N-linked glycoconjugates. After the proteolytic cleavage between the subdomains LG3 and LG4, the LG4–5 tandem domain dissociated from the rest of the G domain. Further, the laminin-5CCG molecule with the 3'LG1–3 chain showed an increased binding affinity for 31 integrin, indicating that proteolytic processing of laminin-5 influences its interaction with 31 integrin.
0021-9258
5184-5193
Kunneken, K.
e0274fe9-8e06-4432-9ba3-ee522766476f
Pohlentz, G.
1d29b4fb-891a-4ac0-a050-0e016b7c271e
Schmidt-Hederich, A.
dcc22f75-ff16-41d5-815f-319c93d41580
Odenthal, U.
a2ffa541-fd5e-4779-b6dd-9e9b0c5b5e05
Smyth, N.
0eba2a40-3b43-4d40-bb64-621bd7e9d505
Peter-Katalinic, J.
0ea408cf-bdb0-4bca-a58f-b265ab89422c
Bruckner, P.
4a2e1674-dc06-4384-951b-45433bc0310a
Eble, J.A.
624c3ba4-46ad-473d-9f39-9c1b8b706e25
Kunneken, K.
e0274fe9-8e06-4432-9ba3-ee522766476f
Pohlentz, G.
1d29b4fb-891a-4ac0-a050-0e016b7c271e
Schmidt-Hederich, A.
dcc22f75-ff16-41d5-815f-319c93d41580
Odenthal, U.
a2ffa541-fd5e-4779-b6dd-9e9b0c5b5e05
Smyth, N.
0eba2a40-3b43-4d40-bb64-621bd7e9d505
Peter-Katalinic, J.
0ea408cf-bdb0-4bca-a58f-b265ab89422c
Bruckner, P.
4a2e1674-dc06-4384-951b-45433bc0310a
Eble, J.A.
624c3ba4-46ad-473d-9f39-9c1b8b706e25

Kunneken, K., Pohlentz, G., Schmidt-Hederich, A., Odenthal, U., Smyth, N., Peter-Katalinic, J., Bruckner, P. and Eble, J.A. (2004) Recombinant human laminin-5 domains - Effects of heterotrimerization, proteolytic processing, and N-glycosylation on alpha(3)beta(1) integrin binding. The Journal of Biological Chemistry, 279 (7), 5184-5193. (doi:10.1074/jbc.M310424200).

Record type: Article

Abstract

Human laminin-5 fragments, comprising the heterotrimeric C-terminal part of the coiled-coil (CC) domain and the globular (G) domain with defined numbers of LG subdomains, were produced recombinantly. The 3' chain with all five LG subdomains was processed proteolytically in a manner similar to the wild-type 3 chain. Conditions were established under which the proteolytic cleavage was either inhibited in cell culture or was brought to completion in vitro. The shorter chains of the laminin-5CCG molecule, 3'and 2', produced in a bacterial expression system associated into heterodimers, which then combined spontaneously with the 3' chains in vitro to form heterotrimeric laminin-5CCG molecules. Only heterotrimeric laminin-5CCG with at least subdomains LG1–3, but not the single chains, supported binding of soluble 31 integrin, proving the coiled-coil domain of laminin-5 to be essential for its interaction with 31 integrin. The N-glycosylation sites in wild-type 3 chain were mapped by mass spectrometry. Their location in a structural model of the LG domain suggested that large regions on both faces of the LG1 and LG2 domains are inaccessible by other proteins. However, neither heterotrimerization nor 31 integrin binding was affected by the loss of N-linked glycoconjugates. After the proteolytic cleavage between the subdomains LG3 and LG4, the LG4–5 tandem domain dissociated from the rest of the G domain. Further, the laminin-5CCG molecule with the 3'LG1–3 chain showed an increased binding affinity for 31 integrin, indicating that proteolytic processing of laminin-5 influences its interaction with 31 integrin.

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Published date: 1 February 2004

Identifiers

Local EPrints ID: 56041
URI: http://eprints.soton.ac.uk/id/eprint/56041
ISSN: 0021-9258
PURE UUID: f0b6a8f6-c947-4e91-a1de-9a1f402b77ca

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Date deposited: 07 Aug 2008
Last modified: 08 Jan 2022 01:12

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Contributors

Author: K. Kunneken
Author: G. Pohlentz
Author: A. Schmidt-Hederich
Author: U. Odenthal
Author: N. Smyth
Author: J. Peter-Katalinic
Author: P. Bruckner
Author: J.A. Eble

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