Laminin-121--recombinant expression and interactions with integrins

Abstract

Laminin-121, previously referred as to laminin-3, was expressed recombinantly in human embryonic kidney (HEK) 293 cells by triple transfection of full-length cDNAs encoding mouse laminin α1, β2 and γ1 chains. The recombinant laminin-121 was purified using Heparin-Sepharose followed by molecular sieve chromatography and shown to be correctly folded by electron microscopy and circular dichroism (CD). The CD spectra of recombinant laminin-121 were very similar to those of laminin-111 isolated from Engelbreth-Holm-Swarm tumor (EHS-laminin) but its T(m) value was smaller than EHS-laminin and recombinant lamnin-111 suggesting that the replacement of the β chain reduced the stability of the coiled-coil structure of laminin-121. Its binding to integrins was compared with EHS-laminin, laminin-3A32 purified from murine epidermal cell line and recombinantly expressed laminins-111, -211 and -221. Laminin-121 showed the highest affinity to α6β1 and α7β1 integrins and furthermore, laminin-121 most effectively supported neurite outgrowth. Together, this suggests that the β2 laminins have higher affinity for integrins than the β1 laminins.

Fig. 1. Immunoblot analysis on the transfected HEK 293 cells

Cell lysates and conditioned media from transfected cells were separated on 3-12% acrylamide gels under reducing conditions and then transfered onto PVDF membranes for the immunodetection with anti-mouse laminin γ1, anti-laminin β1 and anti-laminin β2 chain antibodies. A; γ1-tansfected cells, B; γ1β1-transfected cells, C; γ1β2-transfected cells. Cell; cell lysate, CM; conditioned medium. For B and C, the blots were incubated with anti-β1 or anti-β2 (left blot) prior to the detection of the γ1 chain (right blot).

Fig. 2. SDS-gelelectrophoresis of purified recombinant laminins

EHS- laminin (1), laminin-111 (2), laminin-121 (3), laminin-211 (4) and laminin-221 (5) were separated under non-reducing (A) and reducing conditions (B) and gels were stained with Coommassie Brilliant Blue R-250. The position of the individual laminin chains was independently confirmed by immunoblotting with chain-specific polyclonal antibodies.

Fig. 3. Rotary shadowing electron microscopy of recombinant laminins

(A) laminin-111, (B) laminin-121, (C) laminin-211 and (D) laminin-221. Bar represents 100nm. Each molecule shows typical cross-like structure of laminin having three short arms and one long arm.

Fig. 4. Thermal unfolding of the coiled-coil domain of recombinant laminins-111 (red) and -121 (green) and EHS-laminin (black)

The transition was followed by the change in mean molar ellipticity at 220 nm [Θ]₂₂₀ and the degree of folding F was calculated by F = ([Θ]₂₂₀ - [Θ]_u)/ ([Θ]_f - [Θ]_u) where [Θ]_f and [Θ]_u are the ellipticities of the unfolded and folded state, respectively. Value of [Θ]_f and [Θ]_u showed small linear temperature dependencies, which were evaluated in the temperature range of 20 to 50 °C (folded state) and 70 to 85°C (unfolded state) and subtracted in the evaluation of F.

Fig. 5. Adhesion of α6 integrins transfected cells to recombinant laminins

α6 transfected K562 cells (K562/α6Aβ1) were stimulated with antibody TS 2/16 prior to the addition to 96 well plates coated with different laminins (A). K562/α6β4 cells were used without stimulation (B). EHS-laminin (△), laminin-3A32 (▽), recombinant laminin-111 (●), recombinant laminin-121 (○), recombinant laminin-211(◆) and recombinant laminin-221 (◇). Cells were incubated at 37°C for 60 min and the attached cells were stained with crystal violet. Representative examples of five experiments, each performed in duplicate, are shown.

Fig. 6. Binding of recombinant laminins to immobilized α6β4 integrin

Recombinant soluble α6β4 integrin immobilized onto plastic wells and recombinant laminin-111(●), -121(○), and laminin-3A32 (▽) were used as soluble ligands. A representative of three experiments is shown.

Fig. 7. Adhesion of the X2 variant of α7 integrin transfected K562 cells to recombinant laminins

A, cells were not stimulated with antibody TS 2/16. B, cells were stimulated with the antibody. EHS-laminin (△), recombinant laminin-111 (●), recombinant laminin-121 (○), recombinant laminin-211(◆) and recombinant laminin-221 (◇). Representative examples of five experiments, each performed in duplicate, are shown.

Fig. 8. Laminin isoforms differ in their ability to stimulate neurite outgrowth

Representative pictures of neurite outgrowth after overnight culture on different substrata were shown; uncoated (A), EHS-laminin (B), recombinant laminin-111 (C) recombinant laminin-121 (D), recombinant laminin-211 (E), recombinant laminin-221 (F). Bar represents 200 μm.