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.

Structural Characterization of PTX3 Disulfide Bond Network and Its Multimeric Status in Cumulus Matrix Organization

“…1B). In a previous work, we characterized the recombinant human PTX3 glycosidic moiety and showed it to be mainly composed of monofucosylated disialylated biantennary complex type sugars with minor monofucosylated triantennary and tetra-antennary structures exhibiting different degrees of sialylation (24). According to this analysis, the PTX3 glycosidic moiety accounts for a contribution to the protein monomer molecular mass of about 2.5 kDa.…”

“…Cell culture supernatants were then collected by centrifugation at 1,500 rpm for 10 min and stored at Ϫ80°C. The protein concentration in culture supernatants was measured by a sandwich enzyme-linked immunosorbent assay based on the monoclonal antibody MNB4 and the rabbit polyclonal antibody ␣PTX3pb as reported previously (5,24). Supernatant aliquots containing 20 ng of proteins were analyzed by WB under both denaturing and non-denaturing conditions as described above.…”

“…Like other members of the long pentraxin subfamily, human PTX3 is composed of a pentraxin-like C-terminal domain (amino acids 179 -381), which is homologous to the entire short pentraxin amino acid sequence, and an unrelated N-terminal region (amino acids 18 -178) (22,23). The human protein has a unique N-glycosylation site at Asn 220 that has been described to be fully occupied by complex type oligosaccharides, mainly fucosylated and sialylated biantennary sugars (24). PTX3 glycosylation status affects the protein interaction with C1q thus modulating PTX3-mediated activation of the complement classical pathway (24).…”

“…The human protein has a unique N-glycosylation site at Asn 220 that has been described to be fully occupied by complex type oligosaccharides, mainly fucosylated and sialylated biantennary sugars (24). PTX3 glycosylation status affects the protein interaction with C1q thus modulating PTX3-mediated activation of the complement classical pathway (24). Studies performed with recombinant isolated forms of both PTX3 C-and N-terminal domains provided preliminary information on regions of the molecule involved in ligands recognition.…”

Protective molecules and their cognate antibodies: new players in autoimmunity

“…The binding between C1q and PTX3 occurs on asparagin (Asn 220). The subsequent activation of the complement cascade contributes to the opsonization of pathogens and their phagocytosis by immune cells [92], thus stimulating the innate immune response. As an opsonizing factor, PTX3 directly stimulates phagocytosis of pathogens by macrophages [81,93].…”