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One of the most definitive examples of a vertebrate extraorganismal structural protein can be found in three-spined sticklebacks (Gasterosteus aculeatus). In the breeding male the kidney hypertrophies and synthesizes an adhesive protein called "spiggin," which is secreted into the urinary bladder from where it is employed as a structural thread for nest building. This paper describes the first molecular characterization of spiggin and demonstrates that this adhesive is a protein complex assembled from a potential of three distinct subunits (␣, ␤, and ␥). These subunits arise by alternative splicing, and 11-ketoandrogens induce their expression in stickleback kidneys. Analysis of the predicted amino acid sequence of each subunit reveals a modular organization whose structural elements display a similarity to the multimerization domains found within von Willebrand Factor-related proteins. These results implicate that spiggin utilizes a conserved multimerization mechanism for the formation of a viscous agglutinate from its constituent subunits in the urinary bladders of male sticklebacks. This novel extraorganismal structural protein is therefore ideally suited to its function as an adhesive thread.Extrorganismal proteins are common among invertebrates. Examples range from the fibroin threads forming spider webs and silkworm cocoons (1, 2) to the collagen-based matrixes of the byssus threads of Mytilus and the Drosophila sgs family (3, 4).

Section: Discussionmentioning

confidence: 99%

“…6). All subunits contained a signal peptide with a predicted length of 19 residues at their amino terminus (Table II) (19) and up to four N-glycosylation sites and one O-glycosylation site (Table II) (20). Each subunit exhibited a modular organization and could be divided into different domains.…”

Section: Analysis Of Spiggin Subunit-␣ -␤ and -␥ Proteins-mentioning

confidence: 99%

Molecular Cloning and Characterization of Spiggin

Jones

Lindberg

Jakobsson

et al. 2001

“…The sICAM-1 sequence has nine predicted N-glycosylation sites and seven predicted O-glycosylation sites (45). In SDS-PAGE, the five sICAM-1 glycoforms displayed the expected size differences, with sizes ranging from ϳ80 to 115 kDa (Fig.…”

Section: The Sialic Acid and Galactose Residues That Are Critical Formentioning

confidence: 94%

Sialylated Complex-type N-Glycans Enhance the Signaling Activity of Soluble Intercellular Adhesion Molecule-1 in Mouse Astrocytes

Otto

Schürpf

Folkers

et al. 2004

Intercellular adhesion molecule-1 (ICAM-1) occurs as both a membrane and a soluble, secreted glycoprotein (sICAM-1). ICAM-1 on endothelial cells mediates leukocyte adhesion by binding to leukocyte function associated antigen-1 (LFA-1) and macrophage antigen-1 (Mac-1). Recombinant mouse sICAM-1 induces the production of macrophage inflammatory protein-2 (MIP-2) in mouse astrocytes by a novel LFA-1-and Mac-1-independent mechanism. Here we showed that N-glycan structures of sI-CAM-1 influence its ability to induce MIP-2 production. sICAM-1 expressed in Chinese hamster ovary (CHO) cells was a more potent inducer of MIP-2 production than sI-CAM-1 expressed in HEK 293 cells, suggesting that posttranslational modification of sICAM-1 could influence its signaling activity. To explore the roles of glycosylation in sICAM-1 activity, we expressed sICAM-1 in mutant CHO cell lines differing in glycosylation, including Lec2, Lec8, and Lec1 as well as in CHO cells cultured in the presence of the ␣-mannosidase-I inhibitor kifunensine. Signaling activity of sICAM-1 lacking sialic acid was reduced 3-fold compared with sICAM-1 from CHO cells. The activity of sICAM-1 lacking both sialic acid and galactose was reduced 12-fold, whereas the activity of sICAM-1 carrying only high mannose-type N-glycans was reduced 12-26-fold. sICAM-1 glycoforms carrying truncated glycans retained full ability to bind to LFA-1 on leukocytes. Thus, sialylated and galactosylated complex-type N-glycans strongly enhanced the ability of sICAM-1 to induce MIP-2 production in astrocytes but did not alter its binding to LFA-1 on leukocytes. Glycosylation could therefore serve as a means to regulate specifically the signaling function of sICAM-1 in vivo.

“…1), a feature commonly found with proteins associated with mineralized tissues (1). Strikingly, three potential O-glycosylation sites and 24 phosphorylation sites out of 28, according to NetOGlyc3.1 (43) and NetPhos2.0 (44), respectively, are densely distributed in GYS region II. This may indicate that GYS region II is likely to have a more flexible and complicated folding structure, as it is more sensitive to post-translational modifications than the GYS region I.…”

Section: Characterization Of Deduced Amino Acid Sequence Ofmentioning

confidence: 99%

Cloning and Characterization of Prisilkin-39, a Novel Matrix Protein Serving a Dual Role in the Prismatic Layer Formation from the Oyster Pinctada fucata

Kong

Yan

et al. 2009

Molluscs form their shells out of CaCO 3 and a matrix of biomacromolecules. Understanding the role of matrices may shed some light on the mechanism of biomineralization. Here, a 1401-bp full-length cDNA sequence encoding a novel matrix protein was cloned from the mantle of the bivalve oyster, Pinctada fucata. The deduced protein (Prisilkin-39), which has a molecular mass of 39.3 kDa and an isoelectric point of 8.83, was fully characterized, and its role in biomineralization was demonstrated using both in vivo and in vitro crystal growth assays. Prisilkin-39 is a highly repetitive protein with an unusual composition of Gly, Tyr, and Ser residues. Expression of Prisilkin-39 was localized to columnar epithelial cells of the mantle edge, corresponding to the calcitic prismatic layer formation. Immunostaining in situ and immunodetection in vitro revealed the presence of a characteristic pattern of Prisilkin-39 in the organic sheet and in sheaths around the prisms. Prisilkin-39 binds tightly with chitin, an insoluble polysaccharide that forms the highly structured framework of the shell. Antibody injection in vivo resulted in dramatic morphological deformities in the inner shell surface structure, where large amounts of CaCO 3 were deposited in an uncontrolled manner. Moreover, Prisilkin-39 strictly prohibited the precipitation of aragonite in vitro. Taken together, Prisilkin-39 is the first protein shown to have dual function, involved both in the chitinous framework building and in crystal growth regulation during the prismatic layer mineralization. These observations may extend our view on the rare group of basic matrices and their functions during elaboration of the molluscan shell.