A motif in LILRB2 critical for Angptl2 binding and activation

Abstract

Key Points• A motif in the immunoglobulin domains of LILRB2 is critical to the multimerized Angptl2 binding and signaling activation.• Immobilized anti-LILRB2 supports ex vivo expansion of human cord blood HSCs.A better understanding of the interaction between extrinsic factors and surface receptors on stem cells will greatly benefit stem cell research and applications. Recently, we showed that several angiopoietin-like proteins (Angptls) bind and activate the immune inhibitory receptor human leukocyte immunoglobulin (Ig)-like receptor B2 (LILRB2) to support ex vivo expansion of hematopoietic stem cells (HSCs) and leukemia development. However, the molecular basis for the interaction between Angptls and LILRB2 was unclear. Here, we demonstrate that Angptl2 expressed in mammalian cells forms high-molecular-weight species and that ligand multimerization is required for activation of LILRB2 for downstream signaling. A novel motif in the first and fourth Ig domains of LILRB2 was identified that is necessary for the receptor to be bound and activated by Angptl2. The binding of Angptl2 to LILRB2 is more potent than and not completely overlapped with the binding of another ligand, HLA-G. Immobilized anti-LILRB2 antibodies induce a more potent activation of LILRB2 than Angptl2, and we developed a serum-free culture containing defined cytokines and immobilized anti-LILRB2 that supports a net expansion of repopulating human cord blood HSCs. Our elucidation of the mode of Angptl binding to LILRB2 enabled the development of a new approach for ex vivo expansion of human HSCs. (Blood. 2014;124(6):924-935)

Sorting protein VPS33B regulates exosomal autocrine signaling to mediate hematopoiesis and leukemogenesis

“…This finding is concordant with the observation that HSCs produce high levels of ANGPTLs (53), which may facilitate the sustained activities of HSCs (14,15,54,55). Because the addition of exogenous ANGPTL2 and ANGPTL3 to the culture medium only partially rescued the decreased proliferative abilities of HSCs/LICs, it is possible that other stemness-related secretory proteins may contribute to the loss of function of VPS33B-null HSCs.…”

The Expanding Spectrum of Ligands for Leukocyte Ig-like Receptors

“…In this case, the details of the binding have been characterized. The interaction involves the first and fourth Ig domains of the receptor and maps to short stretches in each with a similar motif GRY/GTY and critical glycine and tyrosine residues (57). The interaction extends to mouse PIRB and can be exploited to promote proliferation and maintenance of human and mouse hematopoietic stem cells in mouse models (56,57).…”

Ex vivo expansion of hematopoietic stem cells

“…In the animal models, Angptls are likely components of the niche of mouse fetal liver and adult HSCs [68,72] and that Angptl1 and 2 are essential to HSC development in zebrafish [155]. We recently showed that leukocyte immunoglobulin-like receptor 2 (LILRB2) is a receptor for multiple Angptls, including GST-Angptl5 [74], and demonstrated that a novel motif in the extracellular domain of LILRB2 mediates Angptl effects [156]. Lin et al [155] demonstrated that, in human CD34 + cells, Angptl2 induces NOTCH activation via the interaction between LILRB2 and NOTCH, resulting in activation of myc targets.…”

“…As immobilized antibodies likely prevent internalization of the ligand LILRB2 (that contains the internalization signal YXXphi [157]), receptor activation is prolonged, and thus the ex vivo expansion of HSCs can be enhanced by the immobilized antibodies. The anti-LILRB2 polyclonal antibodies are more readily expressed and purified and are more stable than Angptls and, importantly, bind and activate LILRB2 with higher efficiency than Angptl2; use of anti-LILRB2 polyclonal antibodies will have advantages in ex vivo HSC expansion systems [156,158].…”

“…We developed a serum-free culture system containing defined cytokines and immobilized anti-LILRB2, which supports a 4.9-fold net expansion of repopulating human cord blood HSCs after 10 d of culture, as determined by NSG transplantation [156]. As immobilized antibodies likely prevent internalization of the ligand LILRB2 (that contains the internalization signal YXXphi [157]), receptor activation is prolonged, and thus the ex vivo expansion of HSCs can be enhanced by the immobilized antibodies.…”