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PRINCIPAL PUBLICATION AND AUTHORS

Intestinal mucin is a chaperone of multivalent copper, N. Reznik (a) A.D. Gallo (b), K.W. Rush (c), G. Javitt (a), Y. Fridmann-Sirkis (a), T. Ilani (a), N.A. Nairner (a), S. Fishilevich (a), D. Gokhman (a), K.N. Chacón (c), K.J. Franz (b), D. Fass (a), Cell 185, 4206-4215 (2022); https:/doi.org/10.1016/j.cell.2022.09.021 (a) Weizmann Institute of Science, Rehovot (Israel) (b) Duke University, Durham (USA) (c) Reed College, Portland (USA)

REFERENCES

[1] G. Javitt et al., Cell 183, 717- 729 (2020).

in the cryo-EM data. This cluster did not appear to be related to the polymerisation mechanism, but the strong conservation of the histidines and methionines in orthologues suggested an important function. The region within the mucin that contained the histidines and methionines was produced separately and shown to specifically bind copper.

Copper is an essential trace metal that is incorporated into enzymes to catalyse redox reactions, due to its ability to exist in the oxidised, Cu(II), and reduced, Cu(I), forms. Copper is physiologically important for mitochondrial respiration and for proper formation of the extracellular matrix. The utility of copper in electron transfer reactions, however, is also a danger. Uncontrolled copper can catalyse the transfer of electrons from electron donors to oxygen, generating reactive oxygen species. For this reason, copper is chaperoned by specialised and well- studied proteins in the circulation and inside cells. Oddly, little thought seems to have been given to the question of how copper is handled in the body before it is transferred to cells and the circulation.

X-ray crystallographic data collected at beamline ID23-2 revealed the geometry of copper binding by the intestinal mucin. Though the cryo-EM structure had shown only a general congregation of the histidines and methionines, the crystal structure revealed a well-resolved coordination site for Cu(II) involving three histidines and one glutamic acid (Figure 14b). In contrast, the methionine residues were not found to be coordinating the copper but rather were lurking about 6 Å away. As methionines are typical Cu(I) ligands, this observation suggested that intestinal mucin has two copper binding sites: one for Cu(II) and another for Cu(I). Indeed, the addition of an electron donor, the antioxidant ascorbic acid (otherwise known as vitamin C) to solutions containing Cu(II)-bound crystals resulted in transfer of the copper to the methionines. Though the crystals were somewhat damaged in this process, diffraction data of sufficient quality could be collected to identify the ~6 Å shift in the peak electron density. Additional experiments, including X-ray absorption of Cu(II)- and Cu(I)-bound protein in solution, confirmed the interpretation of the crystallographic study.

Following the crystallographic analysis, cell culture experiments showed that the copper-binding region of the intestinal mucin protected cells from copper toxicity when added to the culture medium (Figure 14c). However, the presence of the protein in the medium did not prevent the absorption of the low amounts of copper needed to maintain basic physiological processes such as the respiratory chain. These observations are consistent with a protective role for intestinal mucin and raise the question of whether mucin is also involved in helping transfer dietary copper to its transporters in epithelial cell membranes. Among its other important functions, intestinal mucin is now established as an extracellular copper chaperone.