Protein L: an immunoglobulin light chain-binding bacterial protein. Characterization of binding and physicochemical properties (1)
Protein A from staphylococcal strains and protein G from group C and G streptococci are two bacterial cell wall proteins with affinity for the Fc part of mammalian IgG. The anaerobic bacterial strain Peptostreptococcus magnus carries a cell wall protein with affinity for the light chains of human Ig, thus representing a candidate for an Ig-binding protein with such broad binding specificity which is named protein L.
3.78 mg of protein L was solubilized from 3 ml of packed P. magnus, and 2.76 mg (73%) was recovered after purification. Thus, the yield of protein L with this method was 0.92 mg/ml packed bacteria.
The molecular weight of the protein was first estimated to be 95,000 by SDS electrophoresis in the presence of the reducing agent 2-mercaptoethanol. No change of migration was seen in the absence of 2-mercaptoethanol, suggesting that protein L does not contain any intrachain disulfide loops, nor is it composed of disulfide-linked subunits. Protein L was an acidic molecule with a PI of 4.0 for the major 95-kDa band.
Agarose gel electrophoresis shows that protein L migrates with the a-region of human plasma.
A maximum at 276 nm is followed by a slow decrease throughout the UV and visible ranges. The absorption coefficient at 280 nm was 7.9 cm^-1 (1% solution) or 6.0 X 10^4 M^-1 cm^-1 (assuming a Mr of 76,000).
Tryptic fragment 2, except the nonidentifiable amino-terminal residue, could be aligned with a sequence within the W region (positions 506-539, using the numbering of protein G, giving 40% identical residues. Finally, computer prediction of secondary structure suggested more than 50% a-helix for each of the three peptides. All three molecules displayed the lowest binding at 4C and highest bind- ing at 37C. All pH units, except pH 2, allowed binding to occur, and highest binding was achieved at pH7 and 8. The equilibrium constants calcu- latedfromthediagramsarelisted in TableIV.Thevaluesfor IgG, IgA, and IgM are similar,around 10^10 M^-1, and were approximately 7 times higher than for k-chains, 1.5 X 10^9.
The binding of radiolabeled protein L to these three IgGs could not be blocked by a 1000-fold excess of protein G or A but was partly blocked by rabbit anti-mouse light chains. This is consistent with a binding of protein L to the light chains of IgG.
125 I-protein L binds to urinary light chains, which were immobilized directly on the nitrocellulose membranes. When the membranes were incubated with antibodies against a1- microglobulin, another urinary protein, 125 I-protein L was found at the position of this protein, 31 kDa.
Over estimation by SDS electrophoresis of the molecular weight for bacterial cell wall proteins seems to be common. The strength of the binding to k-chains alone was less, approximately one-seventh of the binding to whole Ig, suggesting that the interaction of heavy and light chains in the molecule gives a conformation of the light chains which is more favorable to protein L binding.