The epidermal basement membrane is a composite of separate laminin- or collagen IV-containing networks connected by aggregated perlecan, but not by nidogens

Abstract

The basement membrane between the epidermis and the dermis is indispensable for normal skin functions. It connects, and functionally separates, the epidermis and the dermis. To understand the suprastructural and functional basis of these connections, heterotypic supramolecular aggregates were isolated from the dermal-epidermal junction zone of human skin. Individual suprastructures were separated and purified by immunomagnetic beads, each recognizing a specific, molecular component of the aggregates. The molecular compositions of the suprastructures were determined by immunogold electron microscopy and immunoblotting. A composite of two networks was obtained from fibril-free suspensions by immunobeads recognizing either laminin 332 or collagen IV. After removal of perlecan-containing suprastructures or after enzyme digestion of heparan sulfate chains, a distinct network with a diffuse electron-optical appearance was isolated with magnetic beads coated with antibodies to collagen IV. The second network was more finely grained and comprised laminin 332 and laminins with α5-chains. The core protein of perlecan was an exclusive component of this network whereas its heparan sulfate chains were integrated into the collagen IV-containing network. Nidogens 1 and 2 occurred in both networks but did not form strong molecular cross-bridges. Their incorporation into one network appeared to be masked after their incorporation into the other one. We conclude that the epidermal basement membrane is a composite of two structurally independent networks that are tightly connected in a spot-welding-like manner by perlecan-containing aggregates.

FIGURE 1.

Flow chart representation of isolation protocol of suprastructures from human dermal-epidermal basement membrane. To obtain a postfibrillar extract, dermal fibrils were removed with anti-collagen I immunobeads from the skin extract enriched in components of the epidermal basement membrane (EE). Networks isolated directly from the postfibrillar extract with anti-collagen IV immunobeads or anti-laminin 332 immunobeads are composites comprising both laminins and collagen IV (A). Treatment of the postfibrillar extract with anti-perlecan immunobeads or heparinases leads to the postperlecan or postheparinase extract, respectively. Perlecan-containing network fragments are obtained with anti-perlecan immunobeads (B). Distinct collagen IV- (C and C′) or laminin 332-containing networks (D and D′) could be separated from postperlecan or postheparinase extracts by anti-collagen IV immunobeads or anti-laminin 332 immunobeads. Nidogen-containing suprastructures (E) were removed from the postfibrillar extract by anti-nidogen immunobeads. Composite networks containing laminins and collagen IV (F) were isolated from the postnidogen extract with either anti-collagen IV beads or anti-laminin 332 beads.

FIGURE 2.

Suprastructural networks isolated from postfibrillar extracts contain collagen IV, laminin 332, and perlecan. A and B, immunogold electron microscopy of suprastructural fragments isolated with magnetic immunobeads recognizing collagen IV (A) or laminin 332 (laminin β3-chain) (B). Double immunogold labelings were with antibodies to collagen IV (12-nm gold particles, white arrowheads) and laminin 332 (laminin γ2-chain) (18-nm gold particles, black arrowheads). Note: collagen IV- and laminin 332-containing networks isolated without prior removal of perlecan or enzyme digestion of heparan sulfate chains are connected to each other and are co-isolated. #, electron-dense, amorphous network structures; *, finely grained, honeycomb-like structures. C–F, perlecan-containing suprastructures isolated with immunomagnetic beads recognizing perlecan and analyzed by immunogold electron microscopy. C, double immunogold labeling with antibodies to perlecan core protein (18-nm gold particles, black arrows) and collagen IV (12-nm gold particles, white arrowheads). D, double immunogold labeling with antibodies to perlecan core protein (18-nm gold particles, black arrows) and laminin 332 (laminin γ2-chain) (12-nm gold particles, black arrowheads). E and F, immunogold labeling with antibodies to nidogen 1 (E, white arrows) and nidogen 2 (F, black arrows). E, inset, magnification of perlecan-containing suprastructural fragment. ≫, magnetic anti-perlecan immunobeads. Scale bars, 200 nm; inset scale bar, 50 nm.

FIGURE 3.

Collagen IV- and laminin 332-containing networks of human epidermal basement membrane are connected by perlecan. A and B, immunoblot of collagen IV- and laminin 332-containing networks. Collagen IV- and laminin 332-containing networks were treated with immunomagnetic beads recognizing perlecan (see Fig. 2, C–F). C and D, postperlecan supernatant further separated with magnetic immunobeads into collagen IV- and laminin 332-containing networks that were analyzed by immunoblotting. Immunoblots: collagen IV (A and C) or laminin 332 (B and D). First lanes, EE; second lanes, material attached to immunomagnetic beads recognizing collagen IV (COL IV BP); third lanes, material attached to immunomagnetic beads recognizing laminin 332 (LAM 332 BP). Note: before removal of perlecan, signals for collagen IV (A) and laminin 332 (B) were observed in preparations isolated with anti-collagen IV immunobeads as well as laminin 332 immunobeads. After removal of perlecan, separation of networks is possible, and signals for the α1- and α2-chain of collagen IV are detected only in the collagen IV- (C) and signals for the α3- and γ2-chain of laminin 332 only in the laminin 332-containing networks (D).

FIGURE 4.

Discrete collagen IV- and laminin 332-containing networks of human epidermal basement membrane are separable after removal of perlecan with immunobeads. Immunogold electron microscopy of collagen IV- and laminin 332-containing networks were separated after immunobead removal of perlecan-containing suprastructures. Immunogold labelings were with antibodies to collagen IV (A and B, white arrowheads), laminin 332 (laminin γ2-chain) (C and D, black arrowheads), or the laminin α5-chain (E and F, black arrows). Note: collagen IV- and laminin 332-containing networks have a diffuse, electron-dense, or a finely grained appearance, respectively. Scale bars, 200 nm.

FIGURE 5.

Nidogens are integral parts of both collagen IV- and laminin-containing networks, whereas perlecan core protein occurs solely in the latter. A–F, collagen IV- and laminin 332-containing networks were separated after removal of perlecan-containing suprastructures and analyzed by immunogold electron microscopy. Immunogold labelings were with antibodies to nidogen 1 (A and B, white arrowheads) or nidogen 2 (C and D, black arrowheads). Antibodies to perlecan core protein did not react with this networks (E and F). G and H, alternatively, the postfibrillar supernatant was treated with heparinases I and III, and immunobead separation of collagen IV- (G) and laminin 332-containing networks (H) was analogous. Immunogold labelings were with antibodies to perlecan core protein (G and H, black arrows). ≫ and ≪, magnetic anti-collagen IV immunobeads and anti-laminin 332 immunobeads, respectively. Scale bars, 200 nm.

FIGURE 6.

Nidogens do not contribute to the connection between collagen IV- and laminin-containing networks. Extracts of suprastructural basement membrane fragments were treated sequentially with magnetic immunobeads recognizing nidogen 1 or 2. A and B, isolated structures attached to immunobeads recognizing nidogen 1 and analyzed by immunogold electron microscopy. Immunogold labelings were with antibodies to collagen IV (A, white arrowheads) or laminin 332 (laminin γ2-chain) (B, black arrowheads). A and B insets, magnification of nidogen 1-containing structures. The postnidogen supernatant was further treated with magnetic immunobeads to isolate collagen IV- and laminin 332-containing networks in analogy to the experiments after removal of perlecan and digestion with heparinases I and III, respectively. C–F, isolated collagen IV- (C and E) and laminin 332-containing networks (D and F) analyzed by immunogold electron microscopy. Immunogold labelings were with antibodies to collagen IV (C and D, white arrowheads) or laminin 332 (laminin γ2-chain) (E and F, black arrowheads). Scale bars, 200 nm; inset scale bars, 50 nm.

FIGURE 7.

Nidogens do not contribute to the connection between collagen IV- and laminin-containing networks. Immunoblot analysis of collagen IV- and laminin 332-containing networks isolated after removal of nidogens 1 and 2 is shown. A, collagen IV; B, laminin 332. First lanes 1, EE; second lanes, material attached to immunomagnetic beads recognizing collagen IV (COL IV BP); third lanes, material attached to immunomagnetic beads recognizing laminin 332 (LAM 332 BP).

FIGURE 8.

Model of the suprastructural organization of epidermal basement membrane. Laminin isoforms 511 (A) and 521 (B) co-polymerize by interactions between N-terminal globular LN domains to form a laminin network (D, top view). Lateral aggregation of laminins leads to further polymerization into a heterotypic laminin suprastructure (D) also integrating laminin 332 lacking short arms (C). Formation of networks by the collagen IV isoforms (α1(IV))₂α2(IV) (E) and (α5(IV))₂α6(IV) (F), but not the collagen IV variant α3(IV)α4(IV)α5(IV) requires end-to-end interactions between C-terminal globular NC1 domains and interactions through N-terminal 7S domains (G, top view). Additionally, lateral aggregation and twisting of collagenous domains provide a tightly entangled collagen IV-network (G). Oligomers of the heparan sulfate proteoglycan perlecan (H) connect the two distinct basement membrane networks (J, lateral view) by integrating in a spot-welding manner its core protein into the laminin polymer and its heparan sulfate chains into the collagen IV-network. Nidogens 1 and 2 (I) are integral parts of both networks (J).