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Sulfilimine Cross-Links: A Key to Tissue Evolution

By: Carol Rouzer, Marnett Research Laboratory
Published: December 2, 2013

The stabilization of basement membrane collagen IV by sulfilimine cross-links appears early in evolution, coincident with complex tissue organization.

The epithelial cells that line body cavities and the endothelial cells that line blood vessels are supported by a thin network of fibers known as the basement membrane. This membrane not only provides structural integrity to the epithelium and endothelium, it also serves as a scaffold for the assembly of macromolecules and interacts with the surface receptors of cells in the substructure of the tissue. Consequently the basement membrane is essential to the processes of cellular differentiation and organogenesis. The single major constituent of the basement membrane is collagen IV, which is constructed from a triple helical protomer as its basic building block. Unlike the collagen used to form most connective tissues, such as tendons and ligaments, collagen IV polypeptides retain their C-terminal non-collagenous domain (NC1), which forms an expanded, nonhelical structure at the end of the protomer. To form the network of fibers that make up the basement membrane, the NC1 regions of two protomers aggregate end-to end to yield a hexamer (Figure 1). This hexameric structure is then stabilized by the formation of a sulfilimine bond (S=N) cross-link between methionine-93 (Met93) of one protomer and hydroxylysine 211 (Hyl211) of the opposing protomer in a reaction catalyzed by the enzyme peroxidasin. The sulfilimine bond is crucial to collagen IV strength and stability, leading Vanderbilt Institute of Chemical Biology investigator Billy Hudson and his colleagues to hypothesize that its appearance in evolution would correspond to the development of complex tissues and organs. They now present compelling data in support of that hypothesis [A. L. Fidler et al., (2013) Proc. Natl. Acad. Sci. U.S.A., published online December 16, DOI:10.1073/pnas.1318499111].

Figure 1. Cartoon representation of the molecular structure of the NC1 domains of two collagen trimers joined end-to-end to form a hexamer. Sulfilimine bonds between Met-93 of one monomer and Hyl211 of the opposing monomer stabilize the structure. Image reproduced through the courtesy of Wikimedia Commons under the GNU Free Documentation License.

To test their hypothesis, the researchers selected 30 different species of Metazoa from 11 different phyla spanning the full range of evolutionary diversity. For each species, they investigated the presence or absence of sulfilimine bonds in the animal’s collagen IV by multiple methods. First, they examined the amino acid sequences of the collagen IV monomers looking for the presence of Met93 and Hyl211. Next, they isolated collagen IV from the animal and treated it with collagenase to release the NC1 domain hexamer. Further treatment with SDS caused dissociation of the hexamer into individual monomers, but if sulfilimine cross-links were present, the linked monomers could not dissociate and would be detected as dimers by SDS-PAGE. Finally, mass spectral analysis of tryptic digests of the NC1 hexamers revealed a characteristic pair of peptides linked by the sulfilimine bridge in those collagen IV samples in which the cross-linking was present (Figure 2).

Figure 2. (A) Sulfilimine bonds cross-link the NC1 domain of one chain of a collagen IV triple helical monomer to the NC1 domain of an opposing monomer chain. Treatment with collagenase releases the NC1 hexamer from the rest of the collagen molecule. Denaturation with SDS then separates the subunits. Those joined by a sulfilimine bond remain as dimers, while the unbonded chains separate into monomers. (B) Samples of collagen IV are treated as described in (A). Then, SDS-PAGE detects dimers and monomers in samples containing sulfilimine bonds. (C) Tryptic treatment of collagen IV yields two peptides joined by the sulfilimine bond. Loss of 2 m/z units relative to the mass predicted from the amino acid composition of the peptides corresponds to the double bond formed between the nitrogen and sulfur atoms of the sulfilimine linkage. Reprinted with permission from [A. L. Fidler et al., (2013) Proc. Natl. Acad. Sci. U.S.A., published online December 16, DOI:10.1073/pnas.1318499111]. Copyright 2013, A. L. Fidler, et al.


The investigators discovered that Met93 and Hyl211 were present in all Eumetazoa that they investigated with the exception of one Cnidarian species Hydra magnipapillata. In contrast, these amino acids were not present in the collagen IV of the non-eumetazoan phyla Porifera, Placozoa, and Choanozoa. Consistently, both SDS PAGE and mass spectrometric analysis confirmed the presence of sulfilimine-linked dimers in the collagen IV of the Eumetazoan species in which Met93 and Hyl211 were also present. The results suggested that the appearance of the sulfilimine bond coincided with the divergence of Cnidaria from Porifera. The absence of sulfilimine dimers in non-eumetazoan species was consistent with the absence of organized tissue and organ structures in these very simple animals.

A search of genomic information confirmed that peroxidasin was conserved in all Eumetazoa. In fact, it was also expressed in some non-eumetazoan species. This enzyme, which produces hypohalous acid needed as an oxidant in sulfilimine bond formation, is related to the vertebrate peroxidases myeloperoxidase, eosinophil peroxidase, and lactoperoxidase, all of which have antibacterial functions. Peroxidasin is the only peroxidase expressed in both invertebrates and vertebrates. Its appearance so early in evolution suggests that it may have played a role in a primitive form of innate immunity prior to its function in sulfilimine bond formation.

Prior work had demonstrated that knockout of peroxidasin in the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster results in lethality during early development. The investigators extended these studies by demonstrating the expression of peroxidasin early in the embryonic development of the zebrafish (Danio rerio). They then treated zebrafish embryos with a morpholino oligonucleotide to reduce expression of peroxidasin. At 24 h post fertilization, the treatment resulted in a mixture of normal embryos (20/45), embryos with a curved trunk (21/45), and markedly deformed embryos (4/45). Examination of the collagen IV in the embryos revealed that the curved trunk phenotype was associated with an absence of sulfilimine-linked dimers and an overall reduction in collagen IV (Figure 3). Thus, sulfilimine bond formation is required for normal embryonic development in the zebrafish.


Figure 3. (A) Expression of peroxidasin and the collagen IV monomers increases during the course of zebrafish embryogeneis. Treatment of embryos with control morpholino oligonucleotide (B) resulted in no abnormalities in development, whereas a morpholino oligonucleotide directed against peroxidasin resulted in a range of phenotypes (C) from highly deformed (D) through normal (F). The abnormal phenotype corresponded to an absence of sulfilimine-linked collagen IV dimers and an overall deficiency in collagen IV (G). Reprinted with permission from [A. L. Fidler et al., (2013) Proc. Natl. Acad. Sci. U.S.A., published online December 16, DOI:10.1073/pnas.1318499111]. Copyright 2013, A. L. Fidler, et al.

Together the results confirm that sulfilimine bond formation appeared very early in evolution, coinciding with the development of complex tissue structures. The single puzzling result occurred in the case of the Cnidaria H. magnipapillata. The investigators note that this organism likely lacks sulfilimine bond formation as a result of a secondary gene loss, as has been previously reported in Hydra species. The extracellular matrix of H. magnipapillata appears to be designed more for flexibility than strength, which could explain the absence of sulfilimine bonds in this species.

The investigation of sulfilimine bond formation across so many species required a large effort with contributions from many investigators. Among these was a group of over 80 middle school, high school, undergraduate, and graduate students from the Aspirnaut Program. These students, most from rural areas underrepresented in the science, technology, engineering, and mathematics fields, have benefited from this ambitious program that aims to increase diversity in the sciences by combining outreach to schools with summer research internships and personal mentoring. Started in 2007 by Billy Hudson, his wife, Julie Hudson, sister, Ann Hudson Kincl, and brother, Johnny Hudson, the Asprinaut Program has touched the lives of students in eight states so far. The participation of so many on this ground-breaking project is a reflection of the program’s success and promise for the future.









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