Laminin chain assembly is regulated by specific coiled-coil interactions

Abstract

Laminins are large heterotrimeric, multidomain proteins that play a central role in organising and establishing all basement membranes. Despite a total of 45 potential heterotrimeric chain combinations formed through the coiled-coil domain of the 11 identified laminin chains (alpha1-5, beta1-3, gamma1-3), to date only 15 different laminin isoforms have been reported. This observation raises the question whether laminin assembly is regulated by differential gene expression or specific chain recognition. To address this issue, we here perform a complete analysis of laminin chain assembly and specificity. Using biochemical and biophysical techniques, all possible heterotrimeric combinations from recombinant C-terminal coiled-coil fragments of all chains were analysed. Apart from laminin 323 (alpha3, beta2, gamma3), for which no biochemical evidence of its existence in vivo is available, these experiments confirmed all other known laminin isoforms and identified two novel potential chain combinations, laminins 312 (alpha3, beta1, gamma2) and 422 (alpha4, beta2, gamma4). Our findings contribute to the understanding of basement membrane structure, function and diversity.

Fig. 1

Purified recombinant laminin coiled-coil fragments. SDS–PAGE analysis of individual chains under reducing conditions. (A) Lane 1, α1; lane 2, α2; lane 3, α3; lane 4, α4; lane 5, α5. (B) Lane 1, β1; lane 2, β2; lane 3, β3. (C) Lane 1, γ1; lane 2, γ2; lane 3, γ3. The migration of marker proteins is indicated.

Fig. 2

β and γ Chain complexes. (A) 10% non-denaturing gel showing heterotypic complex formation of selected chain combinations (lane 4, β1γ2; lane 5, β1γ1) in comparison with the corresponding homotypic laminin coiled-coil fragments (lane 1, β1; lane 2, γ1; lane 3, γ2). (B) Summary of heterotypic complex formation of all possible β and γ chain combinations. Complex formation on non-denaturing gels was confirmed by in-gel trypsin digestion and subsequent mass spectroscopy.

Fig. 3

Selected laminin αβγ chain combinations. (A) 10% non-denaturing gel of selected chain combinations representing known laminin isoforms (lane 2, α1β1γ1 complex; lane 3, α2β1γ1 complex; lane 4, α3β1γ1 complex) shown in comparison to the β1γ1 complex (lane 1). α Chains are positively charged and therefore do not enter the gel. (B) 10% non-denaturing gel of αβγ chain combinations of unknown laminin isoforms (lane, 2, α1 + β1γ2; lane 3, α2 + β1γ2; lane 4, α3β1γ2) shown in comparison to the β1γ2 complex (lane 1). (C) 10% non-denaturing gel of αβγ chain combinations of unknown laminin chain combinations (lane, 2, α1 + β2γ2; lane 3, α2 + β2γ2; lane 4, α3 + β2γ2; lane 5, α4β2γ2; lane 6, α5 + β2γ2) shown in comparison to the β1γ2 complex (lane 1). Lane 7, α4 chain.

Fig. 4

Circular Dichroism (CD) spectroscopy measurements on recombinant laminin α3β1γ1 and α3β1γ2 complexes. CD spectra of α3β1γ1 (A) and α3β1γ2 (C) recorded at 4 °C. Thermal unfolding profiles of α3β1γ1 (B) and α3β1γ2 (D) monitored by CD following temperature-induced change of the mean molar residue ellipticity at 222 nm, [Θ]₂₂₂. Inset, qualtitative comparison of the temperature-induced unfolding of β1γ2 and α3β1γ2. All measurements were taken at total chain concentration of 10 μM. All protein samples were in 5 mM sodium phosphate buffer (pH 7.4) containing 150 mM NaCl.

Fig. 5

Electron microscopic analysis of recombinant laminin complexes. TEM micrographs of glycerol-sprayed/rotary metal shadowed α3β1γ1 (A) and α3β1γ2 (B) specimens. Histograms with single Gaussian fit representing the distribution of the molecular length of laminin complexes are shown as insets. One hundred molecules were measured, with values displayed representing the mean and standard deviation of the histogram. Scale bar, 50 nm.