Identification of the NC1 domain of {alpha}3 chain as critical for {alpha}3{alpha}4{alpha}5 type IV collagen network assembly

Abstract

The network organization of type IV collagen consisting of α3, α4, and α5 chains in the glomerular basement membrane (GBM) is speculated to involve interactions of the triple helical and NC1 domain of individual α-chains, but in vivo evidence is lacking. To specifically address the contribution of the NC1 domain in the GBM collagen network organization, we generated a mouse with specific loss of α3NC1 domain while keeping the triple helical α3 chain intact by connecting it to the human α5NC1 domain. The absence of α3NC1 domain leads to the complete loss of the α4 chain. The α3 collagenous domain is incapable of incorporating the α5 chain, resulting in the impaired organization of the α3α4α5 chain-containing network. Although the α5 chain can assemble with the α1, α2, and α6 chains, such assembly is incapable of functionally replacing the α3α4α5 protomer. This novel approach to explore the assembly type IV collagen in vivo offers novel insights in the specific role of the NC1 domain in the assembly and function of GBM during health and disease.

FIGURE 1.

Generation of the A3_mA5_h KI mice. A, construction of gene targeting vector and validation of target insertion is shown. A 4.5-kb fragment for mouse α3 Gly-X-Y collagenous domain was inserted adjunct to a 1.8-kb fragment for human α5NC1 domain before the Neo cassette. A 2.1-kb fragment for mouse α3 3′-UTR was inserted after the Neo cassette. Arrows indicate genotyping primers. TK, thymidine kinase. B, genotyping results from PCR from tail genomic DNA, with a 600-bp PCR product for KI allele and 350-bp PCR product for the wild-type α3 allele. Het, heterozygous. C, primers were designed to amplify a fragment from the cDNA sequence of human α5NC1 domain. No amplification in the WT kidney cDNA (negative control) indicates primer specificity. The 200-bp-expected PCR product was detected in A3_mA5_h heterozygote tail genomic DNA containing human α5NC1 cDNA sequence (positive control) and in A3_mA5_h KI kidney cDNA, indicating human α5NC1 expression in the A3_mA5_h KI mouse. Actin amplification was used for internal control. D–E, in situ hybridization is shown using DIG-labeled human α5NC1-specific probe revealed glomeruli expression in A3_mA5_h KI kidney section (E) but no expression in control WT kidney section (D). Glomeruli are circled; arrows point to human α5NC1 expression. F–M, shown is immunolabeling of kidney tissue sections using anti-human α5NC1 antibody, which reveals positive glomeruli labeling in the KI kidney (L–M) but not in the KO (J–K) and WT (H–I) control or secondary antibody negative control (NC) only (F–G). Glomeruli are circled. Magnification: left panel, ×200; the scale bar indicates 50 μm; right panel, ×630, the scale bar indicates 10 μm.

FIGURE 2.

Mouse kidney type IV collagen immunolabeling. A, immunolabeling of KI kidney frozen section for podocin (red) and human (hu) α5NC1 (green) is shown. DAPI nuclear labeling is in blue. Magnification: ×400. B–G, shown is immunolabeling of WT, KO, and KI frozen sections for α1NC1 (B), α2NC1 (C), α3NC1 (D), α4NC1 (E), α5NC1 (F) and α6NC1 (G). Magnification, ×630. NC, negative control, secondary antibody only. Glomeruli are circled in panels D and E.

FIGURE 3.

The chimeric mouse α3/human α5 chain in the A3_mA5_h KI kidney assembles into protomers associated with type IV collagen network. A, shown is a schematic of ECM proteins extraction. Total kidney ECM was collagenase-digested to obtain type IV collagen NC1 hexamer, which can subsequently be denatured and reduced to monomers and dimers. B, shown are Western blot analyses of kidney ECM proteins extracted from human, WT, KO (ablation of mouse α3NC1 domain), and A3_mA5_h KI mice for α1-α6NC1. C and D, shown are Western blot analyses of kidney ECM proteins extracted from human, WT, A3_mA5_h heterozygous (Het) and KI mice for α3NC1 (C) and human α5NC1 (D). Monomers are detected at ∼30 kDa, and dimers are at ∼60 kDa. E, Western blot analyses of ECM proteins extracted from kidney, lung, and testes of KI mice are shown.

FIGURE 4.

Immunoprecipitation studies. A, the chimeric mouse (ms) α3/human (hu) α5 chain (composed of the mouse α3 7S and collagenous domain and human α5NC1 domain)-containing hexamers were subjected to immunoprecipitation using a specific antibody to human α5NC1 domain. B–F, the bound and unbound proteins were analyzed by Western blot using antibodies anti-human α5NC1 (B), α1NC1 (C), α2NC1 (D), mouse α5NC1 (E), and mouse α6NC1 (F). The supernatant of the immunoprecipitation reaction (unbound) was also analyzed, and total kidney ECM from human and KI kidneys was used for positive control. The blots were loaded as follows: 1, human kidney ECM, immunoprecipitated using anti-human α5NC1 antibody; 2, human kidney ECM, immunoprecipitation beads only; 3, supernatant from human kidney ECM IP using anti-human α5NC1 antibody; 4, supernatant from human kidney ECM IP with beads only; 5, KI kidney ECM immunoprecipitation using anti-human α5NC1 antibody; 6, KI kidney ECM IP with beads only; 7, supernatant from KI kidney ECM IP using anti-human α5NC1 antibody; 8, supernatant from KI kidney ECM IP with beads only; 9, human kidney ECM; 10, KI kidney ECM. D, dimer; M, monomer; Δ, degradation product. G, schematic depiction of type IV collagen protomer assembly and type IV collagen network assembly; lack of α3NC1 domain (chimeric chain) precludes α3α4α5 protomer assembly, and the chimeric chain does not assemble into a α1α2α1/α5α6α5 network assembly but forms a unique type IV collagen network incorporating the chimeric chain, thus, highlighting a role for the 7 S and collagenous domains in network assembly.

FIGURE 5.

A3_mA5_h mice develop progressive glomerulonephritis associated with GBM defects. A–D, shown are histological findings from periodic acid-Schiff (PAS) and Masson-Trichrome (MTS) staining of WT, KO, and KI kidneys (A) and morphometric analyses for glomerular sclerosis (B), tubular atrophy (C), and interstitial fibrosis (D). Magnification: upper panel, ×400; middle panel, ×100; lower panel, ×200. NS, not significant. E, shown are urine albumin/urine creatinine ratio measurements at 8, 12, and 22 weeks of age in WT (8 weeks, n = 8; 12 weeks, n = 7; 22 weeks, n = 8), KO (8 weeks, n = 8; 12 weeks, n = 8; 22 weeks, n = 5), and KI (8 weeks, n = 6; 12 weeks, n = 8; 22 weeks, n = 6) mice. F, shown are serum creatinine measurements (mg/dl) at 8, 12, and 22 weeks of age in WT (8 weeks, n = 8; 12 weeks, n = 7; 22 weeks, n = 3), KO (8 weeks, n = 7; 12 weeks, n = 7; 22 weeks, n = 15), and KI (8 weeks, n = 8; 12 weeks, n = 9; 22 weeks, n = 6) mice. G, shown are survival curve of KI mice (n = 11) and KO mice (n = 8). H–J, shown is an electron microscopy picture of WT (H), KO (I), and KI (J) GBM (underlined). P, podocyte; CL, capillary lumen. *, p < 0.05; τ, KO urine and serum samples were also used in urine albumin/creatinine and serum creatinine measurements in previous publication (26).

FIGURE 6.

Recognition sequences for NC1 interaction and “lock and key” NC1 domain assembly in type IV collagen chain selection. A, shown is a schematic representation of the N- and C-terminal lock-and-key assembly of type IV collagen protomers. In the chimeric human α5-mouse α4-α5 schematic the arrow points to preferred binding site. N, N terminus of the NC1 domain; C, C terminus of the NC1 domain. B, shown is sequence alignment of the human and mouse α3, α4, and α5(IV) NC1 domains. The sequences were aligned using the program ClustalW (34). Hyphens represent gaps inserted for optimum alignment. The standard NC1 numbering scheme is shown in red. Missense mutations associated with Alport syndrome are underlined. Highlighted sequences represent putative coding sequence for binding between NC1 terminals.

FIGURE 7.

Modeling of the type IV collagen protomer assembly. A–E, shown is modeling of the mouse type IV collagen protomer. A, shown is a ribbon representation of the mouse α1α2α1 protomer model. The positions of the secondary structure elements were calculated using the program STRIDE (35). α1_a, α1_b, and α2 NC1 domain chains are colored in brown, blue, and purple, respectively. The secondary structure elements of α1_a NC1 domain are labeled. B, hydrogen bonds between the mouse α1_b and mouse α2 NC1 domains are shown. C, shown are hydrophobic interactions between mouse α1_a and α1_b NC1 domains. D, hydrogen bonds between the mouse α3 and mouse α5 NC1 domains are shown. E, shown are hydrophobic interactions between mouse α4 and human α5NC1 chains in a hypothetical human α5/mouse α4α5(IV) protomer.