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Minicollagen cysteine-rich domains encode distinct modes of polymerization to form stable nematocyst capsules.

Tursch A, Mercadante D, Tennigkeit J, Gräter F, Özbek S - Sci Rep (2016)

Bottom Line: Our combined experimental and computational analyses reveal the cysteines in the C-CRD fold to exhibit a higher structural propensity for disulfide bonding and a faster kinetics of polymerization.During nematocyst maturation, the highly reactive C-CRD is instrumental in efficient cross-linking of minicollagens to form pressure resistant capsules.The higher ratio of C-CRD folding types evidenced in the medusozoan lineage might have fostered the evolution of novel, predatory nematocyst types in cnidarians with a free-swimming medusa stage.

View Article: PubMed Central - PubMed

Affiliation: University of Heidelberg, Centre for Organismal Studies, Department of Molecular Evolution and Genomics, Im Neuenheimer Feld 329, 69120 Heidelberg, Germany.

ABSTRACT
The stinging capsules of cnidarians, nematocysts, function as harpoon-like organelles with unusual biomechanical properties. The nanosecond discharge of the nematocyst requires a dense protein network of the capsule structure withstanding an internal pressure of up to 150 bar. Main components of the capsule are short collagens, so-called minicollagens, that form extended polymers by disulfide reshuffling of their cysteine-rich domains (CRDs). Although CRDs have identical cysteine patterns, they exhibit different structures and disulfide connectivity at minicollagen N and C-termini. We show that the structurally divergent CRDs have different cross-linking potentials in vitro and in vivo. While the C-CRD can participate in several simultaneous intermolecular disulfides and functions as a cystine knot after minicollagen synthesis, the N-CRD is monovalent. Our combined experimental and computational analyses reveal the cysteines in the C-CRD fold to exhibit a higher structural propensity for disulfide bonding and a faster kinetics of polymerization. During nematocyst maturation, the highly reactive C-CRD is instrumental in efficient cross-linking of minicollagens to form pressure resistant capsules. The higher ratio of C-CRD folding types evidenced in the medusozoan lineage might have fostered the evolution of novel, predatory nematocyst types in cnidarians with a free-swimming medusa stage.

No MeSH data available.


Related in: MedlinePlus

Immunocytochemistry for N-CRD and C-CRD antibodies.(A) Animals were stained using NCol-1 antibody (scale bar: 800 μm). (A) Nests of developing nematocytes are only detectable in the body column of Hydra (scale bar: 25 μm). (B) Animals were co-stained with N-CRD (green) and C-CRD (red) antibodies. Immunolocalization showed overlapping and distinct distributions of N-CRD and C-CRD signals in the body column of Hydra. Scale bar: 200 μm. (C) N-CRD and C-CRD signals in nests of differentiating nematocysts in the gastric region. Scale bar: 100 μm. (D,E) Close-up of tentacle region. N-CRD antibody stains mature nematocysts of all types in battery cells (D) while the C-CRD antibody does not show signals in the tentacle region (E). Scale bar: 50 μm. Nuclei were stained using DAPI (blue).
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f5: Immunocytochemistry for N-CRD and C-CRD antibodies.(A) Animals were stained using NCol-1 antibody (scale bar: 800 μm). (A) Nests of developing nematocytes are only detectable in the body column of Hydra (scale bar: 25 μm). (B) Animals were co-stained with N-CRD (green) and C-CRD (red) antibodies. Immunolocalization showed overlapping and distinct distributions of N-CRD and C-CRD signals in the body column of Hydra. Scale bar: 200 μm. (C) N-CRD and C-CRD signals in nests of differentiating nematocysts in the gastric region. Scale bar: 100 μm. (D,E) Close-up of tentacle region. N-CRD antibody stains mature nematocysts of all types in battery cells (D) while the C-CRD antibody does not show signals in the tentacle region (E). Scale bar: 50 μm. Nuclei were stained using DAPI (blue).

Mentions: Antibodies raised against CRDs in various nematocyst proteins have proven highly specific due to the low sequence similarity of the CRDs5101122. To investigate the in vivo assembly dynamics of the two CRDs we have raised polyclonal antibodies against N- and C-CRD peptides of Hydra minicollagen-1 (NCol-1) (Fig. 1A). As shown by Western blotting, the antibodies tested against the KLH-conjugated CRD peptides did not show cross-reactivity for the other CRD sequence or against KLH alone (Fig. S4). Pre-adsorption of the respective antibodies with unconjugated CRD peptides resulted in a complete loss of the signal. Immunofluorescence analysis using polyclonal anti-NCol-1 antibody raised against full-length NCol-123 only stained nests of developing nematocysts in the body column of the animal (Fig. 5A). In these clusters, NCol-1 is visualized in the secretory pathway as well as in the growing nematocyst capsule (Fig. 5A′). Co-staining of Hydra whole mounts using both CRD antibodies revealed only partial overlap of the respective signals (Fig. 5B,C), indicating varying and discrete accessibility of the two domains during nematocyst morphogenesis. Both CRD antibodies stained clusters of developing nematocyst stages in the gastric region (Fig. 5B), but in contrast to anti-C-CRD and anti-NCol-1, anti-N-CRD also reacted with mature capsules in the tentacles (Fig. 5D,E). The notorious lack of antibody staining for mature nematocysts has been explained with capsule wall compaction by polymerization of CRD-containing proteins11131422. We therefore concluded that the N-CRD of NCol-1 is involved in disulfide-linked polymers to a lesser extend than the C-CRD. This finding was confirmed when we analyzed hydra tissue lysate and isolated nematocysts by Western blotting. Although the C-CRD antibody showed a higher reactivity for fully reduced NCol-1 in tissue lysates (Fig. S5A), minicollagen polymers from isolated capsules that were partially solubilized by DTT titration exposed more antigen binding sites at low DTT concentrations (3–6 mM) for the N-CRD antibody than for the C-CRD antibody (Fig. S5B). Nematocysts develop by an intracellular secretion process during which minicollagens and other proteins are transported via the endoplasmic reticulum (ER) and Golgi into the post-Golgi vesicle, in which the nematocyst capsule forms. The base of the capsule forms first and then the tubule forms as an extension at one end of the capsule24. After completion, the tubule invaginates and the capsule wall becomes compacted by polymerization of minicollagens and other CRD-containing proteins5. At higher magnification of morphogenetic stages visualized by both CRD antibodies, NCol-1 is detected exclusively by the N-CRD antibody in the early secretory pathway, before entering the trans Golgi network (TGN) (Fig. 6A). In contrast, the C-CRD signal emerges in vesicles of the TGN where it dominates over the N-CRD signal (Fig. 6A). After secretion into the nematocyst vesicle, the two CRD signals merge at the inner face of the vesicle membrane where the capsule wall is formed (Fig. 6B). During tubule invagination minicollagen import ceases and the secretory pathway close to the nematocyst vesicle forms a halo of residual NCol-1 (Fig. 6B). The absence of a signal for the C-CRD antibody in the early secretory pathway indicates a cross-linking of the NCol-1 molecules during this phase via their C-CRDs. Since most collagens of the extracellular matrix possess a C-terminal trimerization domain, we hypothesized that cross-linking of the C-CRDs in newly synthesized minicollagens might play a role in this process. We concluded that, different from the N-CRD, the C-CRD is engaged in intermolecular cross-links of minicollagen chains, probably acting as a C-terminal collagen trimerization motif. These initial cross-links are apparently resolved in the TGN rendering the C-CRD capable of intermolecular cross-links between different minicollagen trimers during capsule maturation (Fig. 6C).


Minicollagen cysteine-rich domains encode distinct modes of polymerization to form stable nematocyst capsules.

Tursch A, Mercadante D, Tennigkeit J, Gräter F, Özbek S - Sci Rep (2016)

Immunocytochemistry for N-CRD and C-CRD antibodies.(A) Animals were stained using NCol-1 antibody (scale bar: 800 μm). (A) Nests of developing nematocytes are only detectable in the body column of Hydra (scale bar: 25 μm). (B) Animals were co-stained with N-CRD (green) and C-CRD (red) antibodies. Immunolocalization showed overlapping and distinct distributions of N-CRD and C-CRD signals in the body column of Hydra. Scale bar: 200 μm. (C) N-CRD and C-CRD signals in nests of differentiating nematocysts in the gastric region. Scale bar: 100 μm. (D,E) Close-up of tentacle region. N-CRD antibody stains mature nematocysts of all types in battery cells (D) while the C-CRD antibody does not show signals in the tentacle region (E). Scale bar: 50 μm. Nuclei were stained using DAPI (blue).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4863159&req=5

f5: Immunocytochemistry for N-CRD and C-CRD antibodies.(A) Animals were stained using NCol-1 antibody (scale bar: 800 μm). (A) Nests of developing nematocytes are only detectable in the body column of Hydra (scale bar: 25 μm). (B) Animals were co-stained with N-CRD (green) and C-CRD (red) antibodies. Immunolocalization showed overlapping and distinct distributions of N-CRD and C-CRD signals in the body column of Hydra. Scale bar: 200 μm. (C) N-CRD and C-CRD signals in nests of differentiating nematocysts in the gastric region. Scale bar: 100 μm. (D,E) Close-up of tentacle region. N-CRD antibody stains mature nematocysts of all types in battery cells (D) while the C-CRD antibody does not show signals in the tentacle region (E). Scale bar: 50 μm. Nuclei were stained using DAPI (blue).
Mentions: Antibodies raised against CRDs in various nematocyst proteins have proven highly specific due to the low sequence similarity of the CRDs5101122. To investigate the in vivo assembly dynamics of the two CRDs we have raised polyclonal antibodies against N- and C-CRD peptides of Hydra minicollagen-1 (NCol-1) (Fig. 1A). As shown by Western blotting, the antibodies tested against the KLH-conjugated CRD peptides did not show cross-reactivity for the other CRD sequence or against KLH alone (Fig. S4). Pre-adsorption of the respective antibodies with unconjugated CRD peptides resulted in a complete loss of the signal. Immunofluorescence analysis using polyclonal anti-NCol-1 antibody raised against full-length NCol-123 only stained nests of developing nematocysts in the body column of the animal (Fig. 5A). In these clusters, NCol-1 is visualized in the secretory pathway as well as in the growing nematocyst capsule (Fig. 5A′). Co-staining of Hydra whole mounts using both CRD antibodies revealed only partial overlap of the respective signals (Fig. 5B,C), indicating varying and discrete accessibility of the two domains during nematocyst morphogenesis. Both CRD antibodies stained clusters of developing nematocyst stages in the gastric region (Fig. 5B), but in contrast to anti-C-CRD and anti-NCol-1, anti-N-CRD also reacted with mature capsules in the tentacles (Fig. 5D,E). The notorious lack of antibody staining for mature nematocysts has been explained with capsule wall compaction by polymerization of CRD-containing proteins11131422. We therefore concluded that the N-CRD of NCol-1 is involved in disulfide-linked polymers to a lesser extend than the C-CRD. This finding was confirmed when we analyzed hydra tissue lysate and isolated nematocysts by Western blotting. Although the C-CRD antibody showed a higher reactivity for fully reduced NCol-1 in tissue lysates (Fig. S5A), minicollagen polymers from isolated capsules that were partially solubilized by DTT titration exposed more antigen binding sites at low DTT concentrations (3–6 mM) for the N-CRD antibody than for the C-CRD antibody (Fig. S5B). Nematocysts develop by an intracellular secretion process during which minicollagens and other proteins are transported via the endoplasmic reticulum (ER) and Golgi into the post-Golgi vesicle, in which the nematocyst capsule forms. The base of the capsule forms first and then the tubule forms as an extension at one end of the capsule24. After completion, the tubule invaginates and the capsule wall becomes compacted by polymerization of minicollagens and other CRD-containing proteins5. At higher magnification of morphogenetic stages visualized by both CRD antibodies, NCol-1 is detected exclusively by the N-CRD antibody in the early secretory pathway, before entering the trans Golgi network (TGN) (Fig. 6A). In contrast, the C-CRD signal emerges in vesicles of the TGN where it dominates over the N-CRD signal (Fig. 6A). After secretion into the nematocyst vesicle, the two CRD signals merge at the inner face of the vesicle membrane where the capsule wall is formed (Fig. 6B). During tubule invagination minicollagen import ceases and the secretory pathway close to the nematocyst vesicle forms a halo of residual NCol-1 (Fig. 6B). The absence of a signal for the C-CRD antibody in the early secretory pathway indicates a cross-linking of the NCol-1 molecules during this phase via their C-CRDs. Since most collagens of the extracellular matrix possess a C-terminal trimerization domain, we hypothesized that cross-linking of the C-CRDs in newly synthesized minicollagens might play a role in this process. We concluded that, different from the N-CRD, the C-CRD is engaged in intermolecular cross-links of minicollagen chains, probably acting as a C-terminal collagen trimerization motif. These initial cross-links are apparently resolved in the TGN rendering the C-CRD capable of intermolecular cross-links between different minicollagen trimers during capsule maturation (Fig. 6C).

Bottom Line: Our combined experimental and computational analyses reveal the cysteines in the C-CRD fold to exhibit a higher structural propensity for disulfide bonding and a faster kinetics of polymerization.During nematocyst maturation, the highly reactive C-CRD is instrumental in efficient cross-linking of minicollagens to form pressure resistant capsules.The higher ratio of C-CRD folding types evidenced in the medusozoan lineage might have fostered the evolution of novel, predatory nematocyst types in cnidarians with a free-swimming medusa stage.

View Article: PubMed Central - PubMed

Affiliation: University of Heidelberg, Centre for Organismal Studies, Department of Molecular Evolution and Genomics, Im Neuenheimer Feld 329, 69120 Heidelberg, Germany.

ABSTRACT
The stinging capsules of cnidarians, nematocysts, function as harpoon-like organelles with unusual biomechanical properties. The nanosecond discharge of the nematocyst requires a dense protein network of the capsule structure withstanding an internal pressure of up to 150 bar. Main components of the capsule are short collagens, so-called minicollagens, that form extended polymers by disulfide reshuffling of their cysteine-rich domains (CRDs). Although CRDs have identical cysteine patterns, they exhibit different structures and disulfide connectivity at minicollagen N and C-termini. We show that the structurally divergent CRDs have different cross-linking potentials in vitro and in vivo. While the C-CRD can participate in several simultaneous intermolecular disulfides and functions as a cystine knot after minicollagen synthesis, the N-CRD is monovalent. Our combined experimental and computational analyses reveal the cysteines in the C-CRD fold to exhibit a higher structural propensity for disulfide bonding and a faster kinetics of polymerization. During nematocyst maturation, the highly reactive C-CRD is instrumental in efficient cross-linking of minicollagens to form pressure resistant capsules. The higher ratio of C-CRD folding types evidenced in the medusozoan lineage might have fostered the evolution of novel, predatory nematocyst types in cnidarians with a free-swimming medusa stage.

No MeSH data available.


Related in: MedlinePlus