Mapping the binding domain of immunoglobulin light chains for Tamm-Horsfall protein

Abstract

Cast nephropathy, or myeloma kidney, is a potentially reversible cause of chronic renal failure. In this condition, filtered light chains bind to a common site on Tamm-Horsfall protein (THP), which is produced by cells of the thick ascending limb of the loop of HENLE: Subsequent aggregation of these proteins produces casts that obstruct tubule fluid flow and results in renal failure. In the present study, we used the yeast two-hybrid system to determine the site of interaction of light chains with THP. The third complementarity-determining region (CDR3) of both kappa and lambda light chains interacted with THP. These findings were confirmed in a series of competition studies using a synthetic peptide that corresponded to the CDR3 region and purified THP and light chains. Variations in the CDR3 sequence of the light chain affected binding. Thus, the current studies increase our understanding of the process of cast formation and provide an opportunity to develop strategies that may inhibit this interaction and prevent the clinical manifestations of myeloma kidney.

Figure 1.

To determine the THP-binding domain on light chains, a series of mutants of κ (SSH23) and λ (ITPBLL1) light chains was examined. The four framework regions (FR) and three CDRs of the variable subunit of the light chains are shown. The lengths (in bp) of the constructs and interaction of the fusion proteins with THP are displayed.

Figure 2.

Western blots of cytoplasmic extracts of yeast co-expressing fusion proteins that consisted of the pLexA DNA-binding domain and a fragment of human THP and the LexA activation domain fused to κ light chains. Top: The yeast expressed the κ light chain/LexA activation domain proteins (ITPBL5, ITPBL11, BC Syn 9, and SSH23). Bottom: Expression of the THP/pLexA DNA-binding domain fusion protein. The expected contribution of the κ light chain and THP fragments to each of the fusion proteins was ∼23 kd and 29 kd, respectively.

Figure 3.

Top: Biotinylated human THP binds to six different human light chains purified from the urine of patients with renal failure and multiple myeloma. Pre-incubation of THP (0.2 μmol/L) with the CDR3 peptide, MQGTHWPPLT (4 mmol/L), which corresponded to the CDR3 region of SSH23, inhibited binding to the light chains. Bottom: The dose-response effect of this peptide on inhibiting binding of THP to these same light chains bound to microtiter wells. An inhibitory effect was observed even at the lowest concentration (4 μmol/L) of peptide.

Figure 4.

Biotinylated human THP binds to samples of extracts from yeast expressing the various light chain fusion proteins (left). As anticipated from the in vivo studies, biotinylated THP bound to samples containing SSH23, SSH23b, SSH23c, SSH23pep, ITPBLL1, ITPBLL1b, and ITPBLL1c. Binding was inhibited by pre-incubation of the THP, 0.2 μmol/L, with the CDR3 peptide, MQGTHWPPLT, 4 mmol/L (right). Both gels were produced simultaneously and developed using the same film and developing solution.