Chimeric protein identification of dystrophic, Pierson and other laminin polymerization residues

Abstract

Laminin polymerization is a key step of basement membrane self-assembly that depends on the binding of the three different N-terminal globular LN domains. Several mutations in the LN domains cause LAMA2-deficient muscular dystrophy and LAMB2-deficient Pierson syndrome. These mutations may affect polymerization. A novel approach to identify the amino acid residues required for polymerization has been applied to an analysis of these and other laminin LN mutations. The approach utilizes laminin-nidogen chimeric fusion proteins that bind to recombinant non-polymerizing laminins to provide a missing functional LN domain. Single amino acid substitutions introduced into these chimeras were tested to determine if polymerization activity and the ability to assemble on cell surfaces were lost. Several laminin-deficient muscular dystrophy mutations, renal Pierson syndrome mutations, and Drosophila mutations causing defects of heart development were identified as ones causing loss of laminin polymerization. In addition, two novel residues required for polymerization were identified in the laminin γ1 LN domain.

A deletion in the N-terminal polymerizing domain of laminin β2 is a new mouse model of chronic nephrotic syndrome

“…22,23,47,48 The mechanism of reduced LAMB2-Del44 quantity in the GBM was not investigated, leaving the possibility of decreased secretion unsolved, in which case overexpression should improve the Del44 phenotype. However, we note that in vitro data suggested that LAMB2-R246Q is both secretion-and polymerization-defective, 28 and might require some degree of both increased expression and polymerization for complete function. In line with this hypothesis, relatively high LAMB2-R246Q overexpression abrogated aberrant capillary loop GBM LAMB1 and podocyte desmin expression but did not completely prevent proteinuria.…”

“…In an established in vitro assay, laminin polymerization mediates laminin retention on Schwann cells (SCs) via receptors in the cell membrane. 24,[27][28][29][30] Our previous investigation of LAMB2-S83R indicated an approximately 50% reduction in retention of LM-121 heterotrimers containing LAMB2-S83R on SCs relative to wild-type (WT) LM-121. 24 The location of S83R within the Del44 deleted sequence predicts further impairment of laminin polymerization of LM-121 containing LAMB2-Del44.…”

“…LAMB2-Del44 lacks 44 amino acids located within the LN domain that is critical for laminin polymerization (Figure 1). 28,33 The LAMB2-S83R (S80R in humans) and Del44 mutations exhibited approximately 50% and approximately 90% reduced polymerization, respectively, in a cell-based polymerization assay (Figure 2, Supplementary Figure S2). Whereas the S83R mouse required a second hit to produce a phenotype in mice, 24 Del44 mice spontaneously developed albuminuria in the first month ( Figure 3) and nephrotic syndrome between 2 and 3 months (Figure 4).…”

“…22 The potential for rescue of polymerization-defective laminins has been demonstrated in vitro and in vivo with rationally designed laminin short arm-nidogen fusion proteins that rescue polymerization of laminin trimers missing either a, b, or g chain LN domains. 28,49 Such polymerization-focused approaches should also be effective for LN domain missense mutations that hinder polymerization.…”

Linker Protein Repair of LAMA2 Dystrophic Neuromuscular Basement Membranes

“…In a subset of Pierson syndrome patients, the disease results from laminin β2LN mutations that cause a failure of laminin polymerization (Funk et al, 2017). Polymerization can be restored by βLNNd, a homolog of αLNNd that enables polymerization of laminins with βLN mutations (McKee et al, 2018). In Alport syndrome, there is a reduction of disulfide covalent crosslinked collagen due to loss of α3/α4/α5-collagen-IV (Gunwar et al, 1998).…”