Human LYPD8 protein inhibits motility of flagellated bacteria

Abstract

BackgroundWe previously reported that the mouse Ly6/Plaur domain containing 8 (mLypd8), a GPI-anchored protein highly and selectively expressed on colonic epithelia, contributes to segregation of intestinal microbiota and intestinal epithelia and is critical for prevention of intestinal inflammation. In addition, it was found that human LYPD8 (hLYPD8) is expressed in the colonic epithelia and expression of hLYPD8 is reduced in some ulcerative colitis patients. However, the molecular characteristics and functions of hLYPD8 remain unclear. In this study, we generated the hLYPD8 protein and characterized its functions.MethodsTo analyze the characteristics and functions of the hLYPD8 protein, recombinant FLAG-tagged hLYPD8 protein was generated by two kinds of protein expression systems: a mammalian cell expression system and a Pichia pastoris expression system. Recombinant hLYPD8 protein was analyzed by western blot analysis or deglycosylation assay. The effect of the protein on flagellated bacteria was examined by ELISA assay and motility assay using semi-agar plates.ResultshLYPD8 was a highly N-glycosylated GPI-anchored protein, like mLypd8. Moreover, recombinant hLYPD8 protein generated by the Pichia pastoris expression system using the SuperMan5 strain, which enabled production of a large number of proteins with human-like glycosylation, presented the high binding affinity and the motility inhibitory function to flagellated bacteria, such as Proteus mirabilis.ConclusionsThese results demonstrated that hLYPD8 inhibits the motile activity of flagellated bacteria, many of which are involved in intestinal inflammation. The supplementation of recombinant hLYPD8 protein might be a novel therapeutic approach for intestinal inflammation of inflammatory bowel diseases.

Single-Cell Analysis Reveals Unexpected Cellular Changes and Transposon Expression Signatures in the Colonic Epithelium of Treatment-Naïve Adult Crohn’s Disease Patients

“…The latter encodes a protein that protects the gut from microbial invasion and is critical for maintaining barrier integrity and preventing intestinal inflammation. 12 , 13 , 14 LYPD8 is expressed 5-fold greater in CEACAM7 + colonocytes compared with CA1 + late colonocytes, but it is down-regulated significantly in CD in both cell types ( Figure 4 E ). Surprisingly, it also is highly expressed and dramatically reduced in CD in EECs ( Figure 4 E ), although this decrease does not achieve significance likely owing to the very small number of EECs, contributing to low statistical power.…”

“…Although AQP8 has been shown previously to be suppressed in IBD, 10 our study shows that this effect is very likely driven by 1 specific colonocyte subtype. Furthermore, the significant reduction in CD of LYPD8 , which encodes an important antimicrobial factor 12 , 13 , 14 in both CEACAM7 + colonocytes and CA1 + late colonocytes, is a possible indication of the beginning stages of impaired colonocyte contribution to barrier function. We also show that LYPD8 is reduced in expression in EECs from CD patients, which has not been shown previously.…”

Single-cell analysis of colonic epithelium reveals unexpected shifts in cellular composition and molecular phenotype in treatment-naïve adult Crohn’s disease

“…Shared down-regulated genes include CD177 and LYPD8 (Figure 3B), both of which are in the same family of proteins containing the LY6/PLAUR domain. The latter encodes a protein that protects the gut from microbial invasion and is critical for maintaining barrier integrity and preventing intestinal inflammation (Hsu et al, 2017; Okumura et al, 2020; Okumura et al, 2016). LYPD8 is expressed 5-fold greater in CEACAM7 + colonocytes compared to CA1 + late colonocytes, but it is significantly down-regulated in CD in both cell types (Figure 3E).…”

“…Although AQP8 has previously been shown to be suppressed in IBD (Ricanek et al, 2015), our study demonstrates for the first time that this effect is very likely driven by one specific colonocyte subtype. Furthermore, the significant reduction in CD of LYPD8 , which encodes an important anti-microbial factor (Hsu et al, 2017; Okumura et al, 2020; Okumura et al, 2016), in both CEACAM7 + colonocytes and CA1 + late colonocytes, is a possible indication of the beginning stages of impaired colonocyte contribution to barrier function. We also show that LYPD8 is reduced in expression in EECs from CD patients, which has not been demonstrated previously.…”

LYPD8 regulates the proliferation and migration of colorectal cancer cells through inhibiting the secretion of IL‑6 and TNF‑α

“…Recently, a highly glycosylated glycosylphosphatidylinositolanchored protein, known as Ly6/Plaur domain-containing 8 (LYPD8), was identified as a novel molecule contributing to the segregation of intestinal bacteria and intestinal epithelia in the large intestine (1). Lypd8, which is anchored to intestinal epithelial cells in the uppermost epithelial layer, is constitutively shed into the intestinal lumen and preferentially binds to flagellated bacteria from various genera (2). Mice lacking LYPD8 lack the bacteria-free space immediately above the epithelial layer of the colon, indicating that LYPD8 is critical for the segregation of intestinal bacteria and colonic epithelia (3).…”