Beta2-microglobulin isoforms display an heterogeneous affinity for type I collagen

Abstract

It has been claimed that beta2-microglobulin (beta2-m) interacts with type I and type II collagen, and this property has been linked to the tissue specificity of the beta2-m amyloid deposits that target the osteo-articular system. The binding parameters of the interaction between collagen and beta2-m were determined by band shift electrophoresis and surface plasma resonance by using bovine collagen of type I and type II and various isoforms of beta2-m. Wild-type beta2-m binds collagen type I with a Kd of 4.1 x 10(-4) M and type II with 2.3 x 10(-3) M. By the BIAcore system we monitored the binding properties of the conformers of the slow phase of folding of beta2-m. The folding intermediates during the slow phase of folding do not display any significant difference with respect to the binding properties of the fully folded molecule. The affinity of beta2-m truncated at the third N-terminal residue does not differ from that reported for the wild-type protein. Increased affinity for collagen type I is found in the case of N-terminal truncated species lacking of six residues. The Kd of this species is 3.4 x 10 (-5) M at pH 7.4 and its affinity increases to 4.9 x 10(-6) M at pH 6.4. Fluctuations of the affinity caused by beta2-m truncation and pH change can cause modifications of protein concentration in the solvent that surrounds the collagen, and could contribute to generate locally a critical protein concentration able to prime the protein aggregation.

Figure 1.

Electrophoretic mobility of β2-m (1), β2-m/collagen type I (2), and collagen type I (3) analyzed at a concentration of 5 μg/mL and 500 μg/mL for β2-m and collagen, respectively. Afterward, agarose gel electrophoresis proteins were blotted and immunodetected by using an antihuman β2-m polyclonal antibody.

Figure 2.

Overlay of sensorgrams measuring the interaction of β2-m (A) and ΔN6 β2-m (B) at different concentrations with the collagen type I immobilized on a sensor chip CM5 and recorded at pH 6.4 and 25°C. Each sensorgram has been evaluated using the BIA evaluation 3.0 software provided with the system. The arrows show the start and the end of injection, respectively.

Figure 3.

A collagen fiber interacting with β2-m. The actual dimensions of the fiber (blue tube) with respect to a single molecule (dot) are reported in the drawing proportions. The boxed region is magnified on the left to show the details of the collagen microfibrils (dark blue) with respect to a β2-m molecule (yellow-gray). In the figure, a single collagen microfibril, formed by assembly of five triple helix structures, is highlighted in red. The box dimensions on the right and the separation from the fiber surface of the dot inside the box have been deliberately exaggerated with respect to the magnification on the left for sake of clarity.

Figure 4.

CD spectra in the near UV region of native β2-m, β2-m thermally denatured at 90°C, and refolded β2-m 1200 sec after the cooling of the solution at 25°C. The figure also shows (dotted line) the spectrum of the protein before the slow phase of folding.