Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 May;134(5):1313-1322.
doi: 10.1038/jid.2013.492. Epub 2013 Nov 14.

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

Rebecca J Richardson  1 , Jan M Gebauer  2 , Jin-Li Zhang  1 , Birgit Kobbe  2 , Douglas R Keene  3 , Kristina Røkenes Karlsen  4 , Stefânia Richetti  5 , Alexander P Wohl  2 , Gerhard Sengle  2 , Wolfram F Neiss  4 , Mats Paulsson  6 , Matthias Hammerschmidt  7 , Raimund Wagener  8
Affiliations

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

Rebecca J Richardson et al. J Invest Dermatol. 2014 May.

Abstract

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.

PubMed Disclaimer

Conflict of interest statement

Conflict of Interest

The authors state no conflict of interest.

Figures

Figure 1
Figure 1. AMACO expression is affected in zebrafish models of Fraser syndrome
Immunofluorescence analysis of whole-mount zebrafish at 32 hpf reveals that (a) AMACO is strongly expressed in the myosepta and developing caudal fin of wild-type zebrafish, very similar to the expression of Fras1 (inset). (b,c) By contrast, AMACO is completely absent from a zebrafish Fras1 null allele (fras1te262/te262) (b), and partially lost from a hypomorphic Fras1 allele (fras1tm95b/tm95b) which exhibits some residual Fras1 expression (c). (d-f) AMACO expression is normal in null alleles for Frem2a (frem2ata90/ta90; d), Frem1a (frem1atc280b/tc280b; e) and Hemicentin1 (hmcn1tq207/tq207; f). Expression of Fras1 is also normal in each of these alleles (inset in d–f). Scale bar = 500 μm. (g,h) Quantitative AMACO immunoblot analysis (Gebauer et al., 2010) in the zebrafish mutants shown in a–f. In each case a pool of 50 fish of a given genotype was extracted to give sufficient material and to minimize effects of individual variations. In g, Ponceau loading control is shown.
Figure 2
Figure 2. AMACO expression is lost from Fras1 mutant mice
(a-f) Immunofluorescence analysis of the epidermis of E14.5 wild type (a,d), Fras1+/bl (b,e) and Fras1bl/bl (c,f) littermates reveals a complete loss of AMACO expression from the basement membrane of Fras1bl/bl mice (c), whereas laminin expression is normal (f). (g–l) Similar analysis of other regions demonstrates the loss of AMACO from a blister over the eye of an E14.5 Fras1bl/bl embryo (g) whereas laminin expression was still observed under the detached epidermis (j). Strong expression of AMACO was observed surrounding the developing tooth germs of E14.5 wild-type mice (h) but was completely lost from a Fras1bl/bl littermate (i) despite laminin being normal (k,l). Scale bars = 50 μm. For generation and characterization of the mouse AMACO-P3 antibody see Gebauer et al., 2009.
Figure 3
Figure 3. Fras1 and AMACO co-localize at the basement membrane
For immuno-EM analysis, newborn mouse skin was immunolabeled enbloc with antibodies directed against mouse AMACO-P3, and mouse Fras1-CSPG (a,b) or human collagen VII and mouse Fras1-CSPG (c). Secondary antibodies conjugated to gold particles of different size (Fras1: 6 nm; AMACO and collagen VII: 10 nm) were detected at anchoring plaques below the lamina densa (LD, black arrowheads). Often anchoring fibrils (AF, open arrowheads) are seen to intersect the anchoring plaques (AP). Collagen VII (arrows) and Fras1 often occur at opposite sides of anchoring plaques (c). (a and c) shows an overview and (b) shows selected anchoring plaques. The scale bar corresponds to 100 nm in (a and c) and 50 nm in (b).
Figure 4
Figure 4. AMACO and Fras1 interact directly
(a) Domain structures of Fras1 and AMACO. (S) signal peptide, (TM) transmembrane domain, the arrow indicates the furin cleavage site. (b, c) Surface plasmon resonance sensorgrams showing binding of different concentrations of soluble analytes to Fras1 CSPG (b) and AMACO P2 (c) coupled onto a chip. Full-length AMACO interacts with Fras1 CSPG, as does the AMACO P2 fragment. AMACO P1 and AMACO P3 do not interact (b). In the reversed orientation Fras1 CSPG interacts with AMACO P2 (c). All proteins were from mouse.
Figure 5
Figure 5. AMACO deficient zebrafish are phenotypically normal
(a–b) Immunofluorecence analysis with antibodies against the zebrafish proteins (Gebauer et al., 2010; Carney et al. 2010) reveals complete loss of AMACO protein in vwa2 morphant (b), whereas levels of Fras1 are normal (inset in b). (c,d) vwa2 morphants display normal fin morphology at 48 hpf. (e,f) Transmission electron microscopy (TEM) analysis at 48 hpf reveals no defect in the structure of the myosepta in vwa2 morphants (f) when compared to wild-type controls (e). (g,h) No abnormalities can be observed in the craniofacial cartilages or general morphology of vwa2 morphants at 80 hpf (h). My = myosepta; Pq, palatoquadrate; Ch, ceratohyal; Ih, interhyal; Hm, hyomandibular; Sy, symplectic.
Figure 6
Figure 6. vwa2 knockdown in Fras1 hypomorphic zebrafish increases the severity of the phenotype
(a) Chart depicting the relative severity of the blistering phenotype of Fras1 zebrafish injected with a missense morpholino (5mm) or a vwa2 specific MO. In all cases, mutant embryos were derived from heterozygous in-crosses, segregating in a normal Mendelian ratio. Only homozygotes are considered in the chart. Included are representative images of mild, moderate and severe blistering. Severe blistering was determined by a large number of caudal fin blisters (arrowheads) often extending further anterior within the dorsal region of the caudal fin. Associated extensive blistering of the caudal vein region was also observed (arrows). Moderate blistering involved fewer fin tip blisters of variable sizes and less, although always some associated caudal vein blistering. Mild blistering was determined by a small number of fin tip blisters with no associated caudal vein blistering. Compare to b for a representative wild type fin. (b) Immunolocalization of Fras1 (with zebrafish-specific antibody, Carney et al. 2010) and laminin for each condition.
See this image and copyright information in PMC

Comment in

  • A new component of the Fraser complex.
    Hiroyasu S, Jones JCR. Hiroyasu S, et al. J Invest Dermatol. 2014 May;134(5):1192-1193. doi: 10.1038/jid.2013.514. J Invest Dermatol. 2014. PMID: 24732331 Free PMC article.

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alazami AM, Shaheen R, Alzahrani F, et al. FREM1 mutations cause bifid nose, renal agenesis, and anorectal malformations syndrome. Am J Hum Genet. 2009;85:414–18. - PMC - PubMed
    1. Carney TJ, Feitosa NM, Sonntag C, et al. Genetic analysis of fin development in zebrafish identifies furin and hemicentin1 as potential novel fraser syndrome disease genes. PLoS Genet. 2010;6:e1000907. - PMC - PubMed
    1. Dalezios Y, Papasozomenos B, Petrou P, et al. Ultrastructural localization of Fras1 in the sublamina densa of embryonic epithelial basement membranes. Arch Dermatol Res. 2007;299:337–43. - PubMed
    1. Gebauer JM, Karlsen KR, Neiss WF, et al. Expression of the AMACO (VWA2 protein) ortholog in zebrafish. Gene Expr Patterns. 2010;10:53–59. - PubMed
    1. Gebauer JM, Keene DR, Olsen BR, et al. Mouse AMACO, a kidney and skin basement membrane associated molecule that mediates RGD-dependent cell attachment. Matrix Biol. 2009;28:456–62. - PubMed

Publication types

MeSH terms