Structure and function of the long pentraxin PTX3 glycosidic moiety

Fine-tuning of the Interaction with C1q and Complement Activation

Antonio Inforzato, Giuseppe Peri, Andrea Doni, Cecilia Garlanda, Alberto Mantovani, Antonio Bastone, Andrea Carpentieri, Angela Amoresano, Piero Pucci, Anja Roos, Mohamed R. Daha, Silvia Vincenti, Grazia Gallo, Paolo Carminati, Rita De Santis, Giovanni Salvatori

Research output: Contribution to journalArticle

80 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)11540-11551
Number of pages12
JournalBiochemistry
Volume45
Issue number38
DOIs
Publication statusPublished - Sep 26 2006

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Complement C1q
Classical Complement Pathway
Complement Activation
N-Acetylneuraminic Acid
Glycosylation
Tuning
Chemical activation
Sugars
Pattern Recognition Receptors
Glycoside Hydrolases
Innate Immunity
Gel Chromatography
Polysaccharides
Fertility
Digestion
Size exclusion chromatography
Proteins
Stabilization
Kinetics
Fluids

ASJC Scopus subject areas

  • Biochemistry

Cite this

Structure and function of the long pentraxin PTX3 glycosidic moiety : Fine-tuning of the Interaction with C1q and Complement Activation. / Inforzato, Antonio; Peri, Giuseppe; Doni, Andrea; Garlanda, Cecilia; Mantovani, Alberto; Bastone, Antonio; Carpentieri, Andrea; Amoresano, Angela; Pucci, Piero; Roos, Anja; Daha, Mohamed R.; Vincenti, Silvia; Gallo, Grazia; Carminati, Paolo; De Santis, Rita; Salvatori, Giovanni.

In: Biochemistry, Vol. 45, No. 38, 26.09.2006, p. 11540-11551.

Research output: Contribution to journalArticle

Inforzato, A, Peri, G, Doni, A, Garlanda, C, Mantovani, A, Bastone, A, Carpentieri, A, Amoresano, A, Pucci, P, Roos, A, Daha, MR, Vincenti, S, Gallo, G, Carminati, P, De Santis, R & Salvatori, G 2006, 'Structure and function of the long pentraxin PTX3 glycosidic moiety: Fine-tuning of the Interaction with C1q and Complement Activation', Biochemistry, vol. 45, no. 38, pp. 11540-11551. https://doi.org/10.1021/bi0607453
Inforzato, Antonio ; Peri, Giuseppe ; Doni, Andrea ; Garlanda, Cecilia ; Mantovani, Alberto ; Bastone, Antonio ; Carpentieri, Andrea ; Amoresano, Angela ; Pucci, Piero ; Roos, Anja ; Daha, Mohamed R. ; Vincenti, Silvia ; Gallo, Grazia ; Carminati, Paolo ; De Santis, Rita ; Salvatori, Giovanni. / Structure and function of the long pentraxin PTX3 glycosidic moiety : Fine-tuning of the Interaction with C1q and Complement Activation. In: Biochemistry. 2006 ; Vol. 45, No. 38. pp. 11540-11551.
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AU - Garlanda, Cecilia

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AU - Bastone, Antonio

AU - Carpentieri, Andrea

AU - Amoresano, Angela

AU - Pucci, Piero

AU - Roos, Anja

AU - Daha, Mohamed R.

AU - Vincenti, Silvia

AU - Gallo, Grazia

AU - Carminati, Paolo

AU - De Santis, Rita

AU - Salvatori, Giovanni

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N2 - 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.

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