CANX

Calnexin (encoded by the gene CANX, abbreviated as CNX) is an essential molecular chaperone involved in glycoprotein quality control in the endoplasmic reticulum (ER). It plays a critical role in maintaining cellular homeostasis and has been implicated in several diseases, including cancer, neurodegenerative disorders and viral infections. Given its functional importance in protein folding and cellular stress responses, calnexin has emerged as a potential drug target.

NCBI Gene ID: 821

UniProtKB ID: P27824

Structure and Function of Calnexin

Calnexin is a 90 kDa protein characterized by an ER luminal domain, a single transmembrane segment and a cytosolic tail. It is part of the calnexin/calreticulin cycle that assists in the proper folding of nascent glycoproteins. Calnexin specifically recognizes monoglucosylated N-glycans on newly synthesized proteins and retains them in the ER until they reach their correct conformation. Interaction with the protein disulfide isomerase ERp57 further enhances its role in disulfide bond formation and quality control. This chaperone function is critical for cellular integrity and its dysfunction is implicated in several pathological conditions.

Figure 1. The calnexin/calreticulin substrate binding cycle. Proteins targeted to the ER receive N-linked glycans that are transferred by the OST complex to acceptor sites. The first two glucoses are trimmed by glucosidases I and II, leaving a monoglucosylated glycan. In this state, the glycan is a substrate for calnexin and calreticulin. Release from calnexin/calreticulin and trimming of the final glucose by glucosidase II leaves the glycan in a non-glucosylated state. Productive folding and adoption of a native state allows for trafficking of the glycoprotein from the ER. Glycoproteins that do not adopt a native fold can be recognized by the folding sensor UDP-glucose: glycoprotein glucosyltransferase 1 (UGGT1). UGGT1 reglucosylates substrates, allowing for rebinding to calnexin/calreticulin or trimming by glucosidase II. Glycoproteins that continue to non-productively fold can be removed from the calnexin/calreticulin cycle through trimming by mannosidases and targeting to ER associated degradation (ERAD) machinery. (Adams et al., 2019)

Calnexin and Disease Pathogenesis

• Cancer: Calnexin plays an important role in tumorigenesis by influencing the unfolded protein response (UPR) and ER stress pathways. Its overexpression has been observed in various cancers, including lung, breast and liver cancer. Calnexin-mediated modulation of glycoprotein folding contributes to cancer cell survival and metastasis. In addition, calnexin interacts with immune checkpoint proteins to influence tumor immune evasion.
• Neurodegenerative Disorders: Protein misfolding is a hallmark of neurodegenerative diseases such as Alzheimer's and Parkinson's disease. Calnexin dysfunction leads to impaired ER-associated degradation (ERAD), resulting in the accumulation of misfolded proteins and neurotoxicity. Studies have shown that calnexin deficiency exacerbates neurodegenerative phenotypes, while pharmacological modulation of calnexin can attenuate protein aggregation and neuronal damage.
• Viral Infections: Several viruses use calnexin for replication and assembly. Human immunodeficiency virus (HIV), hepatitis C virus (HCV) and coronaviruses use calnexin-mediated glycoprotein folding to enhance viral protein maturation.

Calnexin as a Drug Target

• Immunotherapy Approaches: Given the role of calnexin in immune regulation, monoclonal antibodies and checkpoint inhibitors targeting calnexin-interacting proteins are being explored for cancer immunotherapy. Enhancing immune recognition of calnexin-modified glycoproteins may improve therapeutic outcomes.
• Genetic and RNA-based Therapies: CRISPR-Cas9 and RNA interference (RNAi) technologies have been explored to modulate calnexin expression in disease contexts. Knockdown of calnexin in cancer models has been shown to reduce tumor growth and metastasis, highlighting its therapeutic potential.

Therapeutic Proteins Targeting Calnexin

• Recombinant Human FVIII: Recombinant human Factor VIII (rFVIII) is a bioengineered version of the essential coagulation factor deficient in hemophilia A patients. As a large (∼280 kDa) multi-domain glycoprotein, FVIII undergoes critical post-translational modifications in the endoplasmic reticulum (ER), where the chaperone calnexin plays a key role in its folding and quality control. Calnexin binds to monoglucosylated N-glycans on the B domain of FVIII, preventing premature degradation and promoting proper structural maturation. First-generation rFVIII products demonstrated that optimal calnexin interaction is critical for producing functional, secretion-competent FVIII molecules, although these early versions had shorter half-lives (~12 hours) due to incomplete glycan processing.
• Moroctocog alfa (BDDrFVIII): Moroctocog alfa is a B-domain deleted recombinant FVIII (BDDrFVIII) in which a 906 amino acid segment is replaced by a short 14-residue linker. This modification improves pharmacokinetics while maintaining the dependence on calnexin for folding of the remaining domains (A1-A2 and A3-C1-C2). The truncated design reduces ER retention time but retains critical calnexin-binding N-glycans at Asn41 and Asn239, which remain essential for preventing aggregation. Notably, BDDrFVIII shows improved secretion efficiency while maintaining hemostatic activity, demonstrating how structural optimization can leverage the chaperone function of calnexin without compromising therapeutic efficacy.
• Lonoctocog alfa (Third-Generation rFVIII): Lonoctocog alfa is an example of advanced engineering to optimize calnexin-mediated folding. As a single-chain FVIII (lacking the typical heterodimeric cleavage), it incorporates targeted point mutations (e.g., R1645H) that enhance ER export while retaining all N-glycosylation sites for calnexin binding. This design takes advantage of calnexin's ability to stabilize the A3-C1 interface during folding, a known bottleneck in FVIII production. The result is a molecule with an extended half-life (~19 hours) and reduced immunogenicity due to more consistent glycosylation patterns monitored by calnexin. Its development highlights how understanding chaperone interactions can drive the design of superior hemophilia therapeutics.

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Reference

1. Adams BM, Oster ME, Hebert DN. Protein quality control in the endoplasmic reticulum. Protein J. 2019;38(3):317-329.