Abstract
Cellular uptake of vitamin B12 (cobalamin) requires capture of transcobalamin (TC) from the plasma by CD320, a ubiquitous cell surface receptor of the LDLR family. Here we present the crystal structure of human holo-TC in complex with the extracellular domain of CD320, visualizing the structural basis of the TC-CD320 interaction. The observed interaction chemistry can rationalize the high affinity of CD320 for TC and lack of haptocorrin binding. The in vitro affinity and complex stability of TC-CD320 were quantitated using a solid-phase binding assay and thermostability analysis. Stable complexes with TC were also observed for the disease-causing CD320ΔE88 mutant and for the isolated LDLR-A2 domain. We also determined the structure of the TC-CD320ΔE88 complex, which revealed only minor changes compared with the wild-type complex. Finally, we demonstrate significantly reduced in vitro affinity of TC for CD320 at low pH, recapitulating the proposed ligand release during the endocytic pathway.
Highlights
Cellular uptake of vitamin B12 requires capture of transcobalamin (TC) from the plasma by CD320, a ubiquitous cell surface receptor of the Low-density Lipoprotein Receptor (LDLR) family
Defects at any stage of this process have been found to lead to Cbl deficiency and severe developmental defects in patients11,12. transcobalamin (TC)-bound Cbl is transported into cells by receptor-mediated endocytosis, which requires Ca2 þ -dependent complex formation of TC with its cognate cell surface receptor CD320
CD320 is a member of the Low-density Lipoprotein Receptor (LDLR) family[13,14], a large group of mostly multi-ligand receptors involved in cholesterol uptake and other physiologically essential functions[15,16]
Summary
Cellular uptake of vitamin B12 (cobalamin) requires capture of transcobalamin (TC) from the plasma by CD320, a ubiquitous cell surface receptor of the LDLR family. Several of the proteins facilitating Cbl uptake and transport from saliva to the blood stream have been structurally characterized[19,20,21,22], the final uptake step into cells has not been visualized at a molecular level. Such insight is required to understand the basis of specific TC-Cbl uptake into cells, and to rationalize disease-causing mutations that interfere with this process. The complex with LDLR-A1 appeared less stable on purification, indicating a decreased stability and precluding a detailed thermostability or solid-phase binding analysis
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