Mapping the functional domains of human transcobalamin using monoclonal antibodies

Abstract

Vitamin B 12 (cobalamin, Cbl) is absorbed in the distal ileum with the help of a specific binding protein intrinsic factor (IF) and appears in the circulation bound to another carrier transcobalamin (TC) [1]. Tissue uptake of the TCAECbl complex (holo-TC) is mediated by specific receptors on the surface of the plasma membrane [2]. Holo-TC represents Cbl available for cellular uptake and a decrease in its level would indicate reduced absorption of the vitamin as well as systemic Cbl deficiency. Two new methods have recently been described for the measurement of holo-TC in plasma samples [3,4]. Both methods employ TC-specific antibodies to capture the protein from plasma but lack the specificity needed for direct measurement of holo-TC in serum. The antigenic determinants and the functional domains of TC have not been identified.Cloning [5][6][7] and recent expression of several kindred Cbl-binding proteins [8][9][10][11] Recombinant human transcobalamin (TC) was probed with 17 monoclonal antibodies (mAbs), using surface plasmon resonance measurements. These experiments identified five distinct epitope clusters on the surface of holo-TC. Western blot analysis of the CNBr cleavage fragments of TC allowed us to distribute the epitopes between two regions, which spanned either the second quarter of the TC sequence GQLA…TAAM or the C-terminal peptide LEPA…LVSW(316-427). Proteolytic fragments of TC and the synthetic peptides were used to further specify the epitope map and define the functional domains of TC. Only one antibody showed some interference with cobalamin (Cbl) binding to TC, and the corresponding epitope was situated at the C-terminal stretch TQAS…QLLR(372-399). We explored the receptor-blocking effect of several mAbs and heparin to identify TC domains essential for the interaction between holo-TC and the receptor. The receptor-related epitopes were located within the TC sequence GQLA…HHSV(103-159). The putative heparin-binding site corresponded to a positively charged segment , which also seemed to be necessary for receptor binding. We conclude that conformational changes in TC upon Cbl binding are accompanied by the convergence of multiple domains, and only the assembled conformation of the protein (i.e. holo-TC) has high affinity for the receptor. Co]cyano-Cbl; IF, intrinsic factor; RU, resonance unit; SPR, surface plasmon resonance; TC, transcobalamin; TC p , recombinant human transcobalamin produced in a plant system; TC p11 , TC y31 ,…, the proteolytic fragments of TC p and TC y with the indicated molecular mass; TC y , recombinant human transcobalamin produced in yeast.

Structural basis for mammalian vitamin B 12 transport by transcobalamin

“…Recent mapping of functional domains of human TC by using monoclonal antibodies (20) yielded some information about the receptor-recognition site. Several antibodies and heparin negatively affected TC-receptor interaction.…”

“…Successful isolation (17), nucleotide sequencing (18), and expression in different recombinant systems (19,20) have laid the ground for TC's structure determination, the first for any member of the mammalian Cbl-transporter family. We present the structures of Cbl-complexed recombinant human and bovine TC, as determined by x-ray crystallography, and a detailed description of Cbl binding.…”

Cellular uptake of cobalamin: Transcobalamin and the TCblR/CD320 receptor

“…The folded conformation of TC saturated with Cbl also presents the epitopes for high affinity interaction of the TC-Cbl complex with TCblR. Mapping the functional domains of TC with monoclonal antibodies that block the binding of TC-Cbl with TCblR has identified peptide regions involving amino acids 103 to 159 and the positively charged heparin binding residues 207 to 227 as regions most likely to interact with the receptor (67) (Figure 1). Crystallographic data identifies these domains within the solvent exposed structure of the holo protein involving Tyr 54 – leu 67 (alpha helix 3), Asn 97 –His 133 (a 5), His 149 – 166 (a 7), Lys 189- Thr 207 (end of a helix 8 to 9).…”

New insights into carrier binding and epithelial uptake of the erythropoietic nutrients cobalamin and folate

“…A similar capturing of cobalamin between two rigid domains is well known for several cobalamin-binding enzymes, including the mammalian methionine synthase [13] and methyl-malonyl coenzyme A mutase [14]. Regions for receptormediated binding in the helixes of the amino terminal domain have also been predicted by combining the structural data with the recent mapping of antibodies interfering with receptor binding [15].…”