Characterization of CD200 Ectodomain Shedding

Abstract

We have previously reported the existence of a soluble form of CD200 (sCD200) in human plasma, and found sCD200 to be elevated in the plasma of Chronic Lymphocytic Leukemia (CLL) patients. CLL cells release CD200 at a constitutive level, which could be attenuated partially by ADAM28 silencing. In this study, we further explored mechanisms of CD200 shedding beyond that of ADAM28, and performed biochemical analysis of sCD200 using materials derived from purified CLL cells and Hek293 cells stably transfected with CD200, and antibodies generated specifically against either the extracellular or cytoplasmic regions of CD200. CD200 shedding was enhanced by PMA stimulation, and the loss of cell surface CD200 could be monitored as a reduction in CD200 cell surface expression by flow cytometry, in parallel with an increase in the detection of sCD200 in the supernatant. Western blot analyses and functional studies using CD200R1 expressing Hek293 cells showed that the shed CD200 detected in CLL and Hek293-hCD200 supernatants lacked the cytoplasmic domain of CD200 but retained the functional extracellular domain required for binding to, and phosphorylation of, CD200R. These data confirms that a functionally active CD200 extracellular moiety can be cleaved from the surface of CD200 expressing cells following ectodomain shedding.

Soluble CX3CL1 gene therapy improves cone survival and function in mouse models of retinitis pigmentosa

“…2D). Because soluble variants of both proteins have also been described (37,38), additional AAVs were generated for soluble CD200 (sCD200) and sCX3CL1 using the human Best1 promoter to drive expression in the retinal pigment epithelium (RPE), a cell layer adjacent to the photoreceptors (39) (SI Appendix, Fig. S2).…”

Matrix-metalloproteinases as targets for controlled delivery in cancer: An analysis of upregulation and expression

“…In fact, neither MMP-11 nor MMP-23 have any known ECM substrates, exhibiting only very weak ECM activity[22,23,56,57]. This pleiotropy leads to a myriad of effects in enzyme activation[58], cell signaling regulation[59], chemokine processing[60], and membrane shedding[61], with ultimately larger-scale impacts in embryogenesis and embryo development[62], tissue morphogenesis[63], tissue homeostasis[64], wound healing[65], and inflammation[53]. As expected with the wide range of effects, MMP dyshomeostasis has been associated with several diseases, including arthritis[66], cardiovascular diseases[67], pulmonary diseases[68], and cancer[69].…”

Changes in expression of theCD200 tolerance‐signaling molecule and its receptor (CD200R) by villus trophoblasts during first trimester missed abortion and in chronic histiocytic intervillositis

“…However, a reduction in CD200R1 in villus syncytiotrophoblasts and villus cytotrophoblasts was more dramatic so CD200/CD200R1 ratios increased. 15 But CD200 is also released in soluble form (sCD200), 18 and similarly CD200R1 may be released, binding of sCD200 to cell surface or a soluble CD200R1 could reduce the effective CD200 concentration and action on the usual lymphomyeloid target cells as well as on syncytiotrophoblasts that make β-hCG. 13,14 The CD200 knockout is on a B6 background, such mice are raised in SPF isolator cage facilities, and actual litter size data has not been published.…”

“…But CD200 is also released in soluble form (sCD200), 18 and similarly CD200R1 may be released, binding of sCD200 to cell surface or a soluble CD200R1 could reduce the effective CD200 concentration and action on the usual lymphomyeloid target cells as well as on syncytiotrophoblasts that make β-hCG. The net difference, CD200-CD200R1, could provide a potentially useful estimate of net CD200 activity.…”