The C-Terminal Acidic Region of Calreticulin Mediates Phosphatidylserine Binding and Apoptotic Cell Phagocytosis

Abstract

Calreticulin is a calcium-binding chaperone that is normally localized in the endoplasmic reticulum (ER). Calreticulin is detectable on the surface of apoptotic cell under some apoptosis-inducing conditions, where it promotes phagocytosis and immunogenicity of dying cells. However, the precise mechanism by which calreticulin, a soluble protein, localizes to the outer surface of the plasma membrane of dying cells is unknown, as are the molecular mechanisms that are relevant to calreticulin-induced cellular phagocytosis. Calreticulin comprises three distinct structural domains; a globular domain, an extended arm-like P-domain and a C-terminal acidic region containing multiple low affinity calcium binding sites. We show here that calreticulin, via its C-terminal acidic region, preferentially interacts with phosphatidylserine (PS) compared to other phospholipids, and that this interaction is calcium-dependent. Additionally, exogenous calreticulin binds apoptotic cells via a higher affinity calcium-dependent mode that is acidic-region-dependent. Exogenous calreticulin also binds live cells, including macrophages, via a second, lower affinity P-and globular domain-dependent, but calcium-independent binding mode that likely involves its generic polypeptide-binding site. Truncation constructs lacking the acidic region or arm-like P-domain of calreticulin are impaired in their abilities to induce apoptotic cell phagocytosis by murine peritoneal macrophages. Taken together, the results of this investigation provide the first molecular insights into the phospholipid-binding site of calreticulin as a key anchor point for the cell surface expression of calreticulin on apoptotic cells. These findings also support a role for calreticulin as a PS-bridging molecule that co-operates with other PS-binding factors to promote the phagocytosis of apoptotic cells.

Efferocytosis of dying cells differentially modulate immunological outcomes in tumor microenvironment

“…Consistent with this idea HMGB1 can inhibit PS‐mediated efferocytosis in apoptotic neutrophils . Whether CRT, which binds PS through its C‐terminal acidic region, also blocks PS signaling awaits further investigation. Similarly, studies combining ICD inducers with PS targeting antibodies or AnxV might also have combinatorial value to skew immune responses in favor immunogenicity.…”

“…In addition to their role in ICD, both CRT and HMGB1 have been shown to interact with PS, and therefore it is possible that in addition to their role in inflammatory signaling, exposure of these molecules masks tolerogenic signals of PS‐mediated efferocytosis . Consistent with this idea HMGB1 can inhibit PS‐mediated efferocytosis in apoptotic neutrophils .…”

Structures of parasite calreticulins provide insights into their flexibility and dual carbohydrate/peptide-binding properties

“…Similar to its ER membrane homologue calnexin (CNX) ectodomain, CRT is composed of a globular domain, into which a flexible arm-like P (proline-rich) domain is inserted (Schrag et al, 2001). In addition, a highly charged and flexible C-terminus extension in CRT and CNX can bind numerous calcium ions with low affinity (Michalak et al, 2002;Wijeyesakere et al, 2016). The globular domain is made up of seven concave and six convex -sheets forming a legume lectin fold (Chouquet et al, 2011;Kozlov et al, 2010;Schrag et al, 2001).…”

Calnexin cycle – structural features of the ER chaperone system

“…Likewise, the highly acidic N-terminal and C-terminal regions of calnexin also contain multiple lowaffinity calcium-binding sites [44]. More recent studies demonstrated that the C terminus of calreticulin has a propensity to form a helical structure [45] and its secondary structure gets enhanced in the presence of Ca 2+ ions [45,46]. , and calreticulin 3 (CRT3).…”