Jan M. Gebauer

Molecular basis for the action of the collagen-specific chaperone Hsp47/SERPINH1 and its structure-specific client recognition

Collagen is the most abundant protein in animals and is a major component of the extracellular matrix in tissues such as skin and bone. A distinctive structural feature of all collagen types is a unique triple-helical structure formed by tandem repeats of the consensus sequence Xaa-Yaa-Gly, in which Xaa and Yaa frequently are proline and hydroxyproline, respectively. Hsp47/SERPINH1 is a procollagen-specific molecular chaperone that, unlike other chaperones, specifically recognizes the folded conformation of its client. Reduced functional levels of Hsp47 were reported in severe recessive forms of osteogenesis imperfecta, and homozygous knockout is lethal in mice. Here we present crystal structures of Hsp47 in its free form and in complex with homotrimeric synthetic collagen model peptides, each comprising one Hsp47-binding site represented by an arginine at the Yaa-position of a Xaa-Yaa-Gly triplet. Two of these three binding sites in the triple helix are occupied by Hsp47 molecules, which bind in a head-to-head fashion, thus making extensive contacts with the leading and trailing strands of the collagen triple helix. The important arginine residue within the Xaa-Arg-Gly triplet is recognized by a conserved aspartic acid. The structures explain the stabilization of the triple helix as well as the inhibition of collagen-bundle formation by Hsp47. In addition, we propose a pH-dependent substrate release mechanism based on a cluster of histidine residues.

Genetic Analysis of Fin Development in Zebrafish Identifies Furin and Hemicentin1 as Potential Novel Fraser Syndrome Disease Genes

Using forward genetics, we have identified the genes mutated in two classes of zebrafish fin mutants. The mutants of the first class are characterized by defects in embryonic fin morphogenesis, which are due to mutations in a Laminin subunit or an Integrin alpha receptor, respectively. The mutants of the second class display characteristic blistering underneath the basement membrane of the fin epidermis. Three of them are due to mutations in zebrafish orthologues of FRAS1, FREM1, or FREM2, large basement membrane protein encoding genes that are mutated in mouse bleb mutants and in human patients suffering from Fraser Syndrome, a rare congenital condition characterized by syndactyly and cryptophthalmos. Fin blistering in a fourth group of zebrafish mutants is caused by mutations in Hemicentin1 (Hmcn1), another large extracellular matrix protein the function of which in vertebrates was hitherto unknown. Our mutant and dose-dependent interaction data suggest a potential involvement of Hmcn1 in Fraser complex-dependent basement membrane anchorage. Furthermore, we present biochemical and genetic data suggesting a role for the proprotein convertase FurinA in zebrafish fin development and cell surface shedding of Fras1 and Frem2, thereby allowing proper localization of the proteins within the basement membrane of forming fins. Finally, we identify the extracellular matrix protein Fibrillin2 as an indispensable interaction partner of Hmcn1. Thus we have defined a series of zebrafish mutants modelling Fraser Syndrome and have identified several implicated novel genes that might help to further elucidate the mechanisms of basement membrane anchorage and of the diseases aetiology. In addition, the novel genes might prove helpful to unravel the molecular nature of thus far unresolved cases of the human disease.

O-Glucosylation and O-Fucosylation Occur Together in Close Proximity on the First Epidermal Growth Factor Repeat of AMACO (VWA2 Protein)

AMACO (VWA2 protein) is an extracellular matrix protein of unknown function associated with certain basement membranes in skin, lung, and kidney. AMACO is a member of the von Willebrand factor A-like (VWA) domain containing protein superfamily and in addition to three VWA domains it also contains two epidermal growth factor-like domains. One of these contains the rare, overlapping consensus sequences for both O-glucosylation and O-fucosylation. In earlier studies of other proteins the attachment of either core glucose and fucose moieties or of the respective elongated glycans starting with these monosaccharides has been described. By a detailed mass spectrometric analysis we show that both elongated O-glucosylated (Xyl1–3Xyl1–3Glc) and elongated O-fucosylated glycan chains (NeuAc2–3Gal1–4GlcNAc1–3Fuc) can be attached to AMACO in close proximity on the same epidermal growth factor-like domain. It has been reported that the lack of O-fucosylation can markedly decrease secretion of proteins. However, the secretion of AMACO is not significantly affected when the glycosylation sites are mutated. The number of extracellular matrix proteins carrying the overlapping consensus sequence is very limited and it could be that these modifications have a new, yet unknown function.

Mouse amaco, a kidney and skin basement membrane associated molecule that mediates rgd-dependent cell attachment

The VWA domain-containing extracellular matrix protein AMACO has not been extensively characterized and its function remains unknown. It has been proposed as a potential cancer marker and carries a rare O-glucosylation and O-fucosylation on its first EGF-like domain. AMACO is a basement membrane associated protein, however its exact localization has not been determined. Here we show by immunogold electron microscopy of mouse kidney and skin that AMACO does not occur within the basement membrane but rather subjacent to the basement membrane at its stromal surface. In skin, AMACO often colocalizes with triple-helical domains of collagen VII containing anchoring fibrils as they emerge from the basal lamina. However, the immunogold patterns for AMACO and the C-terminal end of collagen VII show discrete differences, indicating that AMACO and collagen VII do not colocalize at anchoring plaques. In contrast, the localization pattern of AMACO partially overlaps with that for collagen XVIII. In addition, mouse AMACO was shown to support beta1 integrin-mediated adhesion of a keratinocyte-like cell line, HaCaT, and a fibroblast cell line, Wi26, in an RGD-dependent manner, most likely using an RGD-motif near the C-terminus of AMACO. However, the loss of cell adhesion to the C-terminal part of the human AMACO, due to the unique absence of an RGD sequence in the human protein, suggests that cell adhesion is not AMACOs major function.

Structure, evolution and expression of collagen XXVIII: Lessons from the zebrafish.

Collagen XXVIII is the last discovered member of the collagen superfamily and thus has been only sparsely investigated. We studied collagen XXVIII in zebrafish to gain insight into its structure, evolution and expression. In contrast to human and mouse, the zebrafish genome contains four collagen XXVIII genes, col28a1a and -b, and col28a2a and -b. Genomic context and phylogenetic analysis revealed that the a2 branch was lost during evolution of mammals, whereas the duplication of the a1 and a2 branches results from the whole genome duplication in the teleost lineage. Sequence analysis revealed conservation of domain structure and the unique imperfections in the triple helical domain. Two major forms of collagen XXVIII were identified, Col28a1b in adult and Col28a2a in 3-5dpf zebrafish. Composite agarose/polyacrylamide gel electrophoresis revealed that both these chains mainly form dimers of trimers, although Col28a1b appears to be more polydisperse. Homodimers are abundant, although it is possible that complexes consisting of Col28a2a and Col28a1a or -a2b occur. Peptide mass fingerprint analysis revealed that the C-terminal Kunitz domain is often proteolytically processed. In contrast to murine collagen XXVIII, the zebrafish orthologs are widely expressed and not only present in the nervous system. They are differentially expressed in the liver, thymus, muscle, intestine and skin. Altogether our results point to a unique nature of collagen XXVIII within the collagen family.

The pH-dependent Client Release from the Collagen-specific Chaperone HSP47 Is Triggered by a Tandem Histidine Pair

Heat shock protein 47 (HSP47) is an endoplasmic reticulum (ER)-resident collagen-specific chaperone and essential for proper formation of the characteristic collagen triple helix. It preferentially binds to the folded conformation of its clients and accompanies them from the ER to the Golgi compartment, where it releases them and is recycled back to the ER. Unlike other chaperones, the binding and release cycles are not governed by nucleotide exchange and hydrolysis, but presumably the dissociation of the HSP47-procollagen complex is triggered by the lower pH in the Golgi (pH 6.3) compared with the ER (pH 7.4). Histidine residues have been suggested as triggers due to their approximate textbook pKa value of 6.1 for their side chains. We present here an extensive theoretical and experimental study of the 14 histidine residues present in canine HSP47, where we have mutated all histidine residues in the collagen binding interface and additionally all of those that were predicted to undergo a significant change in protonation state between pH 7 and 6. These mutants were characterized by biolayer interferometry for their pH-dependent binding to a collagen model. One mutant (H238N) loses binding, which can be explained by a rearrangement of the Arg222 and Asp385 residues, which are crucial for specific collagen recognition. Most of the other mutants were remarkably silent, but a double mutant with His273 and His274 exchanged for asparagines exhibits a much less pronounced pH dependence of collagen binding. This effect is mainly caused by a lower koff at the low pH values.

AMACO is a component of the basement membrane-associated fraser complex

Fraser syndrome (FS) is a phenotypically variable, autosomal recessive disorder characterized by cryptophthalmus, cutaneous syndactyly, and other malformations resulting from mutations in FRAS1, FREM2, and GRIP1. Transient embryonic epidermal blistering causes the characteristic defects of the disorder. Fras1, Frem1, and Frem2 form the extracellular Fraser complex, which is believed to stabilize the basement membrane. However, several cases of FS could not be attributed to mutations in FRAS1, FREM2, or GRIP1, and FS displays high clinical variability, suggesting that there is an additional genetic, possibly modifying contribution to this disorder. An extracellular matrix protein containing VWA-like domains related to those in matrilins and collagens (AMACO), encoded by the VWA2 gene, has a very similar tissue distribution to the Fraser complex proteins in both mouse and zebrafish. Here, we show that AMACO deposition is lost in Fras1-deficient zebrafish and mice and that Fras1 and AMACO interact directly via their chondroitin sulfate proteoglycan (CSPG) and P2 domains. Knockdown of vwa2, which alone causes no phenotype, enhances the phenotype of hypomorphic Fras1 mutant zebrafish. Together, our data suggest that AMACO represents a member of the Fraser complex.

Expression of the AMACO (VWA2 protein) ortholog in zebrafish.

AMACO is a basement membrane associated protein that belongs to the VWA domain-containing protein superfamily. In addition to three VWA domains it contains two EGF-like domains, a cysteine-rich domain and a unique domain. Mouse AMACO has been partially characterized, but its function remains unknown. The zebrafish genome contains a single AMACO ortholog gene on chromosome 12. The domain structure is completely conserved between zebrafish and mouse and the first EGF-like domain, carrying a rare O-glucosylation and O-fucosylation consensus sequence, has the highest identity at the protein level. RT-PCR shows strongest AMACO expression during development, starting at the 5 somite stage. An antibody specific for zebrafish AMACO detected expression mainly in myosepta but also in skin, pronephros, pituitary gland, otic capsule and gills. In situ hybridization revealed that the muscle precursor cells of the somites express the protein that is laid down in the myosepta.

Microstructure dependent binding of pigment epithelium derived factor (PEDF) to type I collagen fibrils

Pigment epithelium derived factor (PEDF) is a multifunctional extracellular protein. In addition to its known anti-angiogenic and neurotrophic roles in collagen rich tissues, PEDF is thought to be involved in collagen fibril assembly due to its sequence specific binding to the collagen fibril and high expression in regions of active bone formation. In order to image the presence of the protein on the fibrils, PEDF was recombinantly made with a strep tag (strep-PEDF) and then gold nanoparticles conjugated to streptavidin (AuNP) were used as a secondary tag. The gold nanoparticles were detected using phase imaging in tapping mode AFM to image where exogenous PEDF bound in rabbit femur. These findings demonstrate that PEDF binds heterogeneously in cortical rabbit femur. Exogenous PEDF binding was concentrated at areas between microstructures with highly aligned collagen fibrils. Binding was not observed on or within the collagen fibrils themselves.

AMACO is a novel component of the basement membrane associated Fraser complex

Fraser syndrome (FS) is a phenotypically variable, autosomal recessive disorder characterized by cryptophthalmus, cutaneous syndactyly, and other malformations resulting from mutations in FRAS1, FREM2, and GRIP1. Transient embryonic epidermal blistering causes the characteristic defects of the disorder. Fras1, Frem1, and Frem2 form the extracellular Fraser complex, which is believed to stabilize the basement membrane. However, several cases of FS could not be attributed to mutations in FRAS1, FREM2, or GRIP1, and FS displays high clinical variability, suggesting that there is an additional genetic, possibly modifying contribution to this disorder. An extracellular matrix protein containing VWA-like domains related to those in matrilins and collagens (AMACO), encoded by the VWA2 gene, has a very similar tissue distribution to the Fraser complex proteins in both mouse and zebrafish. Here, we show that AMACO deposition is lost in Fras1-deficient zebrafish and mice and that Fras1 and AMACO interact directly via their chondroitin sulfate proteoglycan (CSPG) and P2 domains. Knockdown of vwa2, which alone causes no phenotype, enhances the phenotype of hypomorphic Fras1 mutant zebrafish. Together, our data suggest that AMACO represents a member of the Fraser complex.