Calprotectin

Crystal structure of Mn²⁺ and Ca²⁺ loaded calprotectin, showing two S100A8-S100A9 dimers. The grey and blue chains represent S100A8 and S100A9, respectively. Purple spheres represent Mn²⁺ and green spheres represent Ca²⁺. Only one manganese ion can bind per calprotectin dimer.

The human homologue of calprotectin is a 24 kDa dimer,[6] and is formed by the protein monomers S100A8 (10,835 Da) and S100A9 (13,242 Da).[11][12] The primary structure of calprotectin can vary between species. For instance, the mouse homologue of S100A8 is 10,295 Da,[13] while the S100A9 homologue is 13,049 Da.[14] Early size exclusion chromatography experiments incorrectly indicated that calprotectin had a molecular mass of 36.5 kDa;[2][8] occasionally this value is used in contemporary literature. Calprotectin S100A8-S100A9 dimers can non-covalently pair with one another to form 48 kDa tetramers.

Metal binding

Calprotectin has a high affinity for calcium, zinc, iron, and manganese.[7][8][15][3] Each of S100A8 and S100A9 contain two EF-hand type Ca²⁺ binding sites,[6][9] and calprotectin is able to bind a total of four calcium ions per dimer or eight calcium ions per tetramer.[16] Calcium binding induces a conformational change in the complex that improves its affinity for transition metals, and promotes tetramer formation.[2][6] A maximum of two transition metal ions may bind to each calprotectin S100A8-S100A9 dimer.[6]

A calprotectin dimer can bind only one manganese or iron ion with high affinity, and it can do this only in the presence of calcium.[6][17][3] Zinc can bind at two sites within the calprotectin dimer, and this can occur in the absence of calcium.[2] Calcium, however, improves calprotectin's affinity for zinc.[6] While calprotectin metal binding occurs at the interface of S100A9 and S100A8 monomers, the independent monomers have some capacity for zinc binding, and may contribute to zinc homeostasis within mammals.[2][11][12]

His₆ coordination of Mn²⁺ in calprotectin. S100A8 histidine residues are coloured grey, S100A9 histidine residues are coloured purple.

The first of the two calprotectin metal binding sites consists of a His₃Asp motif, with S100A8 contributing two histidine ligands (His83 and His87), and S100A9 contributing a histidine and an aspartic acid ligand (His20 and Asp30).[6] The second site can coordinate metals through a tetra-histidine (His₄) or a hexa-histidine (His₆) binding motif. In the case of His₄ binding, S100A8 coordinates through both His17 and His27 while S100A9 coordinates through His91 and His95.[6] In hexa-histidine binding two further histidine residues, His103 and His105, are recruited from the C-terminal end of S100A9 to enable octahedral coordination of the transition metal.[6] Manganese or iron are bound by the calprotectin dimer at this His₆ site.[6][3] Zinc can be bound to either of the sites that form at the interface between S100A8 and S100A9 monomers.[6][17]

Calprotectin constitutes up to 60% of soluble protein content in the cytosol of neutrophil granulocytes,[2][7][8] and it can be found at a lower concentration in monocytes, macrophages, and squamous epithelial cells.[2][7][8] Calprotectin enters into pus and abscess fluid during neutrophil cell death, along with other antimicrobial proteins.[2]

Mammalian cells secrete calprotectin during the inflammatory response. Plasma calprotectin is elevated in persons with metabolic syndrome, a disease characterized by chronic inflammation.[18] Calprotectin is secreted in the mouth during inflammation of the gingiva and during oral candidiasis infection.[19][20] People who have mutations in the calprotectin gene appear susceptible to serious gum infections.[19] Manganese sequestration by calprotectin is likely important during lung inflammation.[4] The exact mechanism by which S100A8 and S100A9 is secreted by mammalian cells during inflammation remains unknown.[9]

Transition metals are essential to the survival of all organisms.[21] Mammals strictly limit metal availability as a part of the innate immune system, and this helps prevent infection by microbes and fungi.[21] Calprotectin was first described in the 1980s as a mammalian antimicrobial protein that acts through the sequestration of zinc.[1][2][6] It is now known that calprotectin also has antibacterial and antifungal properties that arise from its ability to sequester manganese and iron.[4][6][3] Calprotectin is the only known antimicrobial agent that acts through manganese sequestration.[6]

Calprotectin becomes available in the intestinal lumen via leukocyte shedding,[1] active secretion,[2][8] cell disturbance, and cell death.[1][8] This results in elevated faecal calprotectin levels, which can be detected in the stool.[1][8] Elevated faecal calprotectin levels therefore indicate migration of neutrophils into the intestinal mucosa, which occurs during intestinal inflammation.[1][8][15] As people with active inflammatory bowel diseases (IBD) such as ulcerative colitis or Crohn disease have as much as a 10-fold increase in faecal calprotectin levels,[7] the measurement of faecal calprotectin can serve as a biochemical test for these diseases.

Although a relatively new test, faecal calprotectin is regularly used as an indicator for IBD during treatment and as a diagnostic marker.[10] Faecal calprotectin tests can also function in distinguishing patients with irritable bowel syndrome from those with IBD.[1][8] Calprotectin is useful as a marker, as it is resistant to enzymatic degradation, and can be easily measured in faeces.[22] Although faecal calprotectin correlates significantly with disease activity in people with confirmed IBD,[23] elevated faecal calprotectin can be a false-positive indicator of IBD under some conditions. Importantly, intake of proton pump inhibitor is associated with significantly elevated calprotectin values.[24] Furthermore, positive faecal calprotectin does not help in localizing IBD, or in distinguishing ulcerative colitis from Crohn's disease.[1] Faecal calprotectin can also indicate other gastrointestinal conditions such as colorectal cancer, gastroenteritis, and food intolerance.[1] Calprotectin levels vary depending on age, comorbidity, and may vary day-to-day within individuals.[1] Faecal calprotectin could be used as a preliminary screen in otherwise functional patients suspected of having IBD, or as a means of following mucosal healing.[1] The potential for using faecal calprotectin in this way is debated, however, and cut-off levels have not been agreed upon.[1]

• Zinc in biology
• Manganese in biology
• Nutritional role of calcium
• Bioinorganic Chemistry