Vertebrate extracellular matrix protein hemicentin-1 interacts physically and genetically with basement membrane protein nidogen-2

Abstract

Hemicentins are large proteins of the extracellular matrix that belong to the fibulin family and play pivotal roles during development and homeostasis of a variety of invertebrate and vertebrate tissues. However, bona fide interaction partners of hemicentins have not been described as yet. Here, applying surface plasmon resonance spectroscopy and co-immunoprecipitation, we identify the basement membrane protein nidogen-2 (NID2) as a binding partner of mammalian hemicentin-1 (HMCN1), in line with the formerly described essential role of mouse HMCN1 for basement membrane integrity. HMCN1 binds to the same protein domain of NID2 (G2) as formerly shown for laminins, but with an approximately ten-fold lower affinity and in a competitive manner. Immunofluorescence and immunogold labellings reveal that HMCN1/Hmcn1 is localized closely attached to basement membranes and in partial overlap with NID2/Nid2a in different tissues of mouse and zebrafish. Genetic knockout and antisense-mediated knockdown studies in zebrafish further show that loss of Nid2a leads to similar defects in fin fold morphogenesis as loss of Laminin-α5 (Lama5) or Hmcn1. Combined partial loss-of-function studies further indicate that nid2a genetically interacts with both hmcn1 and lama5. Together, this suggests that despite their mutually exclusive physical binding, hemicentins, nidogens and laminins tightly cooperate and support each other during formation, maintenance and function of basement membranes to confer tissue linkage.

Hemicentin-mediated type IV collagen assembly strengthens juxtaposed basement membrane linkage

“…Given the critical role of type IV collagen in mediating B-LINK maintenance, we wanted to determine how hemicentin promotes collagen assembly. Hemicentin is not known to bind to type IV collagen ( Zhang et al, 2021 Preprint ), so we hypothesized that hemicentin might recruit another protein that mediates collagen assembly. We reasoned that loss of this protein would result in a similar phenotype to type IV collagen loss and thus compiled a list of 403 genes using Wormbase (see Materials and methods) whose RNAi knockdown or genetic loss leads to the Rup phenotype ( Table S7 ).…”

“…Close association of hemicentin with fibulins and collagens has been observed in both C. elegans and vertebrates ( Feitosa et al, 2012 ; Lin et al, 2020 ; Muriel et al, 2005 ). The physical-binding partners of hemicentins, however, are poorly understood ( Zhang et al, 2021 Preprint ). Studies in C. elegans have shown that hemicentin promotes the assembly of fibuilin-1 at several extracellular sites and requires EGF repeats 4 and 5 of fibulin-1, suggestive of a direct interaction ( Muriel et al, 2005 , 2012 ).…”

Hemicentin mediated type IV collagen assembly strengthens juxtaposed basement membrane linkage

“…Close association of hemicentin with fibulins and collagens have been observed in both C. elegans and vertebrates (Feitosa et al, 2012;Lin et al, 2020;Muriel et al, 2005). The physical binding partners of hemicentins, however, are poorly understood (Zhang et al, 2021). Studies in C. elegans have shown hemicentin promotes the assembly of fibuilin-1 at several extracellular sites and requires EGF repeats 4 and 5 of fibulin-1, suggestive of a direct interaction (Muriel et al, 2005(Muriel et al, , 2012.…”