title: Structure and function of the long pentraxin PTX3 glycosidic moiety: fine-tuning of the interaction with C1q and complement activation.
abstract: The prototypic long pentraxin PTX3 is a unique fluid-phase pattern recognition receptor that plays a nonredundant role in innate immunity and female fertility. The PTX3 C-terminal domain is required for C1q recognition and complement activation and contains a single N-glycosylation site on Asn 220. In the present study, we characterized the structure of the human PTX3 glycosidic moiety and investigated its relevance in C1q interaction and activation of the complement classical pathway. By specific endo and exoglycosidases digestion and direct mass spectrometric analysis, we found that both recombinant and naturally occurring PTX3 were N-linked to fucosylated and sialylated complex-type sugars. Interestingly, glycans showed heterogeneity mainly in the relative amount of bi, tri, and tetrantennary structures depending on the cell type and inflammatory stimulus. Enzymatic removal of sialic acid or the entire glycosidic moiety equally enhanced PTX3 binding to C1q compared to that in the native protein, thus indicating that glycosylation substantially contributes to modulate PTX3/C1q interaction and that sialic acid is the main determinant of this contribution. BIAcore kinetic measurements returned decreasing K(off) values as sugars were removed, pointing to a stabilization of the PTX3/C1q complex. No major rearrangement of PTX3 quaternary structure was observed after desialylation or deglycosylation as established by size exclusion chromatography. Consistent with C1q binding, PTX3 desialylation enhanced the activation of the classical complement pathway, as assessed by C4 and C3 deposition. In conclusion, our results provided evidence of an involvement of the PTX3 sugar moiety in C1q recognition and complement activation.
authors.affiliation: Department of Biology, University Tor Vergata, Rome, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: Inforzato
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Peri
authors.forename: Andrea
authors.initials: A
authors.lastname: Doni
authors.forename: Cecilia
authors.initials: C
authors.lastname: Garlanda
authors.forename: Alberto
authors.initials: A
authors.lastname: Mantovani
authors.forename: Antonio
authors.initials: A
authors.lastname: Bastone
authors.forename: Andrea
authors.initials: A
authors.lastname: Carpentieri
authors.forename: Angela
authors.initials: A
authors.lastname: Amoresano
authors.forename: Piero
authors.initials: P
authors.lastname: Pucci
authors.forename: Anja
authors.initials: A
authors.lastname: Roos
authors.forename: Mohamed R
authors.initials: MR
authors.lastname: Daha
authors.forename: Silvia
authors.initials: S
authors.lastname: Vincenti
authors.forename: Grazia
authors.initials: G
authors.lastname: Gallo
authors.forename: Paolo
authors.initials: P
authors.lastname: Carminati
authors.forename: Rita
authors.initials: R
authors.lastname: De Santis
authors.forename: Giovanni
authors.initials: G
authors.lastname: Salvatori
mesh_terms: D000818:Animals; D002097:C-Reactive Protein / Q000737:chemistry* / Q000378:metabolism*; D016466:CHO Cells; D002850:Chromatography, Gel; D003167:Complement Activation / Q000276:immunology*; D015922:Complement C1q / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D006026:Glycoside Hydrolases / Q000378:metabolism; D006027:Glycosides / Q000737:chemistry* / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D008958:Models, Molecular; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D009844:Oligosaccharides / Q000032:analysis / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D000683:Serum Amyloid P-Component / Q000737:chemistry* / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D000099153:Pentraxins
chemical_list: D002097:C-Reactive Protein; D015922:Complement C1q; D006026:Glycoside Hydrolases; D006027:Glycosides; D019158:N-Acetylneuraminic Acid; D009844:Oligosaccharides; D011994:Recombinant Proteins; D000683:Serum Amyloid P-Component; D000099153:Pentraxins