The Biophysical Interaction of the Danger-Associated Molecular Pattern (DAMP) Calreticulin with the Pattern-Associated Molecular Pattern (PAMP) Lipopolysaccharide.

Edwin Bremer,Valerie Wiersma,Rekha Panchal,Chih-Yuan Chiang,Chinaza Egbuta,Paul Eggleton,Leslie Gold,Trefa Abdullah Norman,Unnati Pandya

doi:10.3390/ijms20020408

Abstract

The endoplasmic reticulum (ER) chaperone protein, calreticulin (CRT), is essential for proper glycoprotein folding and maintaining cellular calcium homeostasis. During ER stress, CRT is overexpressed as part of the unfolded protein response (UPR). In addition, CRT can be released as a damage-associated molecular pattern (DAMP) molecule that may interact with pathogen-associated molecular patterns (PAMPs) during the innate immune response. One such PAMP is lipopolysaccharide (LPS), a component of the gram-negative bacterial cell wall. In this report, we show that recombinant and native human placental CRT strongly interacts with LPS in solution, solid phase, and the surface of gram-negative and gram-positive bacteria. Furthermore, LPS induces oilgomerization of CRT with a disappearance of the monomeric form. The application of recombinant CRT (rCRT) to size exclusion and anion exchange chromatography shows an atypical heterogeneous elution profile, indicating that LPS affects the conformation and ionic charge of CRT. Interestingly, LPS bound to CRT is detected in sera of bronchiectasis patients with chronic bacterial infections. By ELISA, rCRT dose-dependently bound to solid phase LPS via the N- and C-domain globular head region of CRT and the C-domain alone. The specific interaction of CRT with LPS may be important in PAMP innate immunity.

Highlights

Calreticulin (CRT) is a highly conserved embryonically lethal endoplasmic reticulum (ER) protein that is essential for quality control of protein folding and intracellular calcium homeostasis [1]
To determine the amounts of LPS, a universal contaminant in bacterially expressed protein, the Limulus Amebocyte Lysate (LAL) assay, which recognizes the carbohydrate part of LPS, was used to test for possible contaminating endotoxin in the CRT preparations
An important point is that both yeast and native human CRT isolated from placenta and non-bacterial systems contained similar amounts of LPS that incurred during their respective purification procedures

Summary

Introduction

Calreticulin (CRT) is a highly conserved embryonically lethal endoplasmic reticulum (ER) protein that is essential for quality control of protein folding and intracellular calcium homeostasis [1]. As a cell protection mechanism, CRT is involved in the unfolded protein response (UPR) [5,6,7] involving degradation of misfolded proteins or halting translation. A major role for CRT in the innate and adaptive immune response is exemplified by its requirement for phagocytosis and MHC Class I antigen processing by antigen presenting cells (APCs), respectively [8,9,10]. CRT was shown to be a C1q receptor on the cell surface of early apoptotic cells that bound to the globular region of C1q, thereby inducing phagocytosis followed by an immunogenic response, including cytokine release [4,18,19,20,21,22]

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Results

Conclusion