title: Insect Cell Glycosylation and Its Impact on the Functionality of a Recombinant Intracrystalline Nacre Protein, AP24.
abstract: The impacts of glycosylation on biomineralization protein function are largely unknown. This is certainly true for the mollusk shell, where glycosylated intracrystalline proteins such as AP24 (Haliotis rufescens) exist but their functions and the role of glycosylation remain elusive. To assess the effect of glycosylation on protein function, we expressed two recombinant variants of AP24: an unglycosylated bacteria-expressed version (rAP24N) and a glycosylated insect cell-expressed version (rAP24G). Our findings indicate that rAP24G is expressed as a single polypeptide containing variations in glycosylation that create microheterogeneity in rAP24G molecular masses. These post-translational modifications incorporate O- and N-glycans and anionic monosialylated and bisialylated, and monosulfated and bisulfated monosaccharides on the protein molecules. AFM and DLS experiments confirm that both rAP24N and rAP24G aggregate to form protein phases, with rAP24N exhibiting a higher degree of aggregation, compared to rAP24G. With regard to functionality, we observe that both recombinant proteins exhibit similar behavior within in vitro calcium carbonate mineralization assays and potentiometric titrations. However, rAP24G modifies crystal growth directions and is a stronger nucleation inhibitor, whereas rAP24N exhibits higher mineral phase stabilization and nanoparticle containment. We believe that the post-translational addition of anionic groups (via sialylation and sulfation), along with modifications to the protein surface topology, may explain the changes in glycosylated rAP24G aggregation and mineralization behavior, relative to rAP24N.
authors.affiliation: Laboratory for Chemical Physics, Division of Basic Sciences and Center for Skeletal Biology, New York University , 345 E. 24th Street, New York, New York 10010, United States.
authors.forename: Eric P
authors.initials: EP
authors.lastname: Chang
authors.affiliation: Laboratory for Chemical Physics, Division of Basic Sciences and Center for Skeletal Biology, New York University , 345 E. 24th Street, New York, New York 10010, United States.
authors.forename: Iva
authors.initials: I
authors.lastname: Perovic
authors.affiliation: Department of Chemistry, Physical Chemistry, Universität Konstanz , Universitätstrasse 10, Konstanz D-78457, Germany.
authors.forename: Ashit
authors.initials: A
authors.lastname: Rao
authors.affiliation: Department of Chemistry, Physical Chemistry, Universität Konstanz , Universitätstrasse 10, Konstanz D-78457, Germany.
authors.forename: Helmut
authors.initials: H
authors.lastname: Cölfen
authors.affiliation: Laboratory for Chemical Physics, Division of Basic Sciences and Center for Skeletal Biology, New York University , 345 E. 24th Street, New York, New York 10010, United States.
authors.forename: John Spencer
authors.initials: JS
authors.lastname: Evans
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002113:Calcification, Physiologic; D019295:Computational Biology; D004926:Escherichia coli; D049851:Gastropoda / Q000737:chemistry* / Q000648:ultrastructure; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D008970:Molecular Weight; D060734:Nacre / Q000737:chemistry*; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D066329:Protein Aggregates; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012596:Scleroproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: D006023:Glycoproteins; D060734:Nacre; D011134:Polysaccharides; D066329:Protein Aggregates; D011994:Recombinant Proteins; D012596:Scleroproteins