Protein L from Peptostreptococcus magnus Binds to the K Light Chain Variable Domain (1)
Proteins A and G interact primarily with the Fc region; the weak binding to the Fab region is also due to heavy chain interaction. More recently, another bacterial cell wall protein was identified, protein L, derived from Peptostreptococcus magnus, the anaerobic bacterial species that, in contrast to proteins A and G, binds humanIg molecules, regardless of heavy chain class, through interaction with Jg light chains, specifically, k-type molecules.
The effect of reduction of the intrachain disulfide bonds and alkylation of the cysteinyl residues was analyzed. Reduction of protein Mal with dithioerythritol in the presence of 6 M guanidine HCl and alkylation with iodoacetamide resulted in a complete loss of the binding to 125I-protein L, as determined on nitrocellulose membranes. Omission of the alkylation step, i.e. only exposure to the reducing agent in the denaturing solvent followed by immobilization and rinsing of the membranes, or reduction under nondenaturing conditions followed by alkylation, did not result in a loss of the binding to protein L.
Trypsin digestion for 1 h of K-chain Mal and subsequent ion-exchange chromatography yielded two components, 10-kDa, and 25-kDa. The 10-kDa protein represented the VI. domain as judged by its molecular mass and NH2-terminal sequence identity with the native protein.
The 10-kDa VL-related Mal fragment was shown by slot- blotting and SPRIA to bind 125I-labeled protein L. The binding affinity of the VL fragment for protein L was comparable to that of the intact light chain. Based on the observation that, at temperatures >55C, light chain cleavage by pepsin yields different-sized VL-related fragments and an intact CL domain.
A schematic representation of the amino acid composition of the intactK chain Mal, the various enzymatically produced fragments of K chain Mal, and the synthetic 28-residue (~3 kDa) VL peptide is depicted. The reactivitywith protein L of these components and their relationship to the overallstructureofthelightchainmoleculearealsoindicated.
ProteinL does not block the antigen binding activity of IgG molecules. The most common amino acid occurring at each position in the VL domain of kI, kII, kIII, and kIVproteins. The shaded areas highlight amino acids that are identical in the frameworks of all four subgroups or only in the VI, VIII, and VIv subgroups. Three of these amino acid sub- stitutions (positions 18,44,and 74) are shifts in charge. It strands the four-stranded sheet, thus not excluding the possibility that the binding of protein L requires the spatial proximity of more than one region on the portion of KI, KIII,and KIV light chain molecules.
Protein L itself, or Protein L-expressing peptostreptococci, can release histamine from mast cells and basophils.