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J. Biol. Chem., Vol. 283, Issue 17, 11714-11720, April 25, 2008

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Artificial Laminin Polymers Assembled in Acidic pH Mimic Basement Membrane Organization^*

Madalena Martins Sant'Ana Barroso¹, Elisabete Freire², Gabriel S. C. S. Limaverde, Gustavo Miranda Rocha, Evander J. O. Batista³, Gilberto Weissmüller, Leonardo Rodrigues Andrade, and Tatiana Coelho-Sampaio⁴

From the Institute of Biomedical Sciences and the Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, RJ 21941-590, Brazil

Natural laminin matrices are formed on cell membranes by a cooperative process involving laminin self-polymerization and binding to cognate cellular receptors. In a cell-free system, laminin can self-polymerize, given that a minimal critical concentration is achieved. We have previously described that pH acidification renders self-polymerization independent of protein concentration. Here we studied the ultrastructure of acid-induced laminin polymers using electron and atomic force microscopies. Polymers presented the overall appearance of natural matrices and could be described as homogeneous polygonal sheets, presenting struts of 21 ± 5 and 86 ± 3 nm of height, which approximately correspond to the sizes of the short and the long arms of the molecule, respectively. The addition of fragment E3 (the distal two domains of the long arm) did not affect the polymerization in solution nor the formation of adsorbed matrices. On the other hand, the addition of fragment E1', which contains two intact short arms, completely disrupted polymerization. These results indicate that acid-induced polymers, like natural ones, involve only interactions between the short arms. The electrostatic surface map of laminin 1 LG4-5 shows that acidification renders the distal end in the long arms exclusively positive, precluding homophylic interactions between them. Therefore, acidification reproduces in vitro, and at a physiological protein concentration, what receptor interaction does in the cellular context, namely, it prevents the long arm from disturbing formation of the homogeneous matrix involving the short arms only. We propose that acid-induced polymers are the best tool to study cellular response to laminin in the future.

Received for publication, November 13, 2007 , and in revised form, January 23, 2008.

^* This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico Grant 552301/2005-1 and Fundação de Amparo à Pesquisa do Rio de Janeiro Grant E-26/170952/2004. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental figures.

¹ Recipient of a Ph. D. fellowship from Consaelho Nacional de Desenvolvimento Científico e Tecnológico.

² Present address: Faculdade de Tecnologia e Ciências, Ave. Luís Viana Filho 8812, Salvador, BA 41741-590, Brazil.

³ Present address: Laboratory of Protozoology, Federal University of Pará, Belém, PA 66055-240, Brazil.

⁴ To whom correspondence should be addressed. Tel.: 5521-2562-6476; Fax: 5521-2562-6483; E-mail: tcsampaio{at}histo.ufrj.br.

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