Abstract
Human sialic acid-binding immunoglobulin-like lectin 14 (Siglec-14) is a glycan-recognition protein that is expressed on myeloid cells, recognizes bacterial pathogens, and elicits pro-inflammatory responses. Although Siglec-14 is a transmembrane protein, a soluble form of Siglec-14 is also present in human blood. However, the mechanism that generates soluble Siglec-14 and what role this protein form may play remain unknown. Here, investigating the generation and function of soluble Siglec-14, we found that soluble Siglec-14 is derived from an alternatively spliced mRNA that retains intron 5, containing a termination codon and thus preventing the translation of exon 6, which encodes Siglec-14's transmembrane domain. We also note that the translated segment in intron 5 encodes a unique C-terminal 7-amino acid extension, which allowed the specific antibody-mediated detection of this isoform in human blood. Moreover, soluble Siglec-14 dose-dependently suppressed pro-inflammatory responses of myeloid cells that expressed membrane-bound Siglec-14, likely by interfering with the interaction between membrane-bound Siglec-14 and Toll-like receptor 2 on the cell surface. We also found that intron 5 contains a G-rich segment that assumes an RNA tertiary structure called a G-quadruplex, which may regulate the efficiency of intron 5 splicing. Taken together, we propose that soluble Siglec-14 suppresses pro-inflammatory responses triggered by membrane-bound Siglec-14.
Highlights
Human sialic acid– binding immunoglobulin-like lectin 14 (Siglec-14) is a glycan-recognition protein that is expressed on myeloid cells, recognizes bacterial pathogens, and elicits proinflammatory responses
We have previously shown that the expression of Siglec-14 on THP-1 cells enhances pro-inflammatory responses elicited by nontypeable Haemophilus influenzae (NTHi) [19]
We demonstrated that alternative splicing generates sSiglec-14, and a sSiglec-14 isoform with a unique C-terminal heptapeptide is detectable in human sera
Summary
A soluble form of Siglec-14 may be generated by proteolysis of a membrane-bound form of Siglec-14 or by alternative mRNA splicing. We attempted to identify the structural element of sSiglec-14 that is required for its anti-inflammatory activity by introducing a mutation at the arginine residue that is required for sialic acid recognition (R119A) or by truncating the C-terminal heptapeptide (⌬C) Both variants suppressed IL-8 production to similar extents as the native form (Fig. 5B). This result implies that neither C-terminal heptapeptide nor sialic acid recognition is essential for the anti-inflammatory effect of sSiglec-14. We confirmed that a blocking antibody against TLR2 suppressed the Pam3CSK4-induced production of IL-8 and found that the addition of sSiglec-14 further suppressed IL-8 production to some extent (Fig. 8C; the anti-TLR2 antibody was used at nonsaturating concentration) Taken together, these results support our model that sSiglec-14 blocks production of pro-inflammatory cytokines/chemokines by interfering with the cis-interaction between mSiglec-14 and TLR2. These results imply that the G-rich segment in intron 5 suppresses the splicing of intron 5, the effect of this element alone may be relatively small
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
