Equilibrium and Kinetic Analyses of the Interactions between Vitamin B12 Binding Proteins and Cobalamins by Surface Plasmon Resonance

Cannon, Michelle J.; Myszka, David G.; Bagnato, Joshua D.; Alpers, David H.; West, F. G.; Grissom, Charles B.

doi:10.1006/abio.2002.5647

Cited by 30 publications

(30 citation statements)

References 36 publications

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“…Upon binding to IF, the solvent accessibility area of Cbl is reduced to Ϸ19% compared with Ϸ7% in TC (23). This difference does not seem to influence the ability of Cbl to bind to these proteins, as shown by recent studies involving binding of fluorescent analogues and surface plasmon resonance, which show that the affinity of Cbl is similar for all three Cbl-binding proteins (25,26). However, the absence of a His coordination bond and wide binding site in IF might allow Cbl to move in and out of IF freely compared with TC.…”

Section: Resultsmentioning

confidence: 92%

Crystal structure of human intrinsic factor: Cobalamin complex at 2.6-Å resolution

Mathews¹,

Gordon

Chen³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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The structure of intrinsic factor (IF) in complex with cobalamin (Cbl) was determined at 2.6-Å resolution. The overall fold of the molecule is that of an ␣6/␣6 barrel. It is a two-domain protein, and the Cbl is bound at the interface of the domains in a base-on conformation. Surprisingly, two full-length molecules, each comprising an ␣-and a ␤-domain and one Cbl, and two truncated molecules with only an ␣-domain are present in the same asymmetric unit. The environment around Cbl is dominated by uncharged residues, and the sixth coordinate position of Co 2؉ is empty. A detailed comparison between the IF-B12 complex and another Cbl transport protein complex, trans-Cbl-B12, has been made. The pH effect on the binding of Cbl analogues in transport proteins is analyzed. A possible basis for the lack of interchangeability of human and rat IF receptors is presented.5] is a water-soluble vitamin and is essential for the growth and development of all mammals, including humans. Cbl is a member of the corrinoid series of cobalt-containing compounds and is distinguished from some other members of this group by possessing a nucleotide side chain terminating in dimethylbenzimidazole (1, 2). It serves as a key enzymatic cofactor for a number of methyltransferase and mutase reactions occurring in nature (3). In mammals, it contributes the prosthetic group for two important enzymes, methionine synthase (a methyltransferase) and methylmalonylCoA mutase (1, 2, 4).In mammals, three proteins are involved in the uptake, transport, and storage of Cbl. These are gastric intrinsic factor (IF), trans-Cbl II (TC), and haptocorrin (HC). These are immunologically distinct proteins with a protein core of Ϸ46 kDa. IF and HC are heavily glycosylated, but TC is not (5, 6). In humans, the genes for IF and HC are located in chromosome 22 (7,8), whereas the gene for TC is located in chromosome 11 (9). Human IF contains 399-aa residues plus Ϸ15% carbohydrate, giving it a molecular mass of Ϸ60 kDa, as observed by gel filtration (10-13). All three proteins promote Cbl entry through endocytosis involving distinct cell surface receptors (5,(14)(15)(16)(17)(18)(19)(20).Dietary Cbl is bound first to HC in the gastric lumen but is transferred to IF in the duodenum after degradation of HC by pancreatic proteases. IF is responsible for transit of Cbl through the small intestine and delivery of it to the endothelial cells that line the ileum. The IF-Cbl complex is then recognized by the IF-Cbl receptor, cubilin (CUB), located on the luminal side of the intestinal mucosal cells, and mediates its internalization. Lysosomal degradation of the internalized IF-Cbl complex releases Cbl, which is then able to bind to TC, probably within the enterocyte. The TC-Cbl complex is then translocated across the basolateral membrane and released into the circulation, to be taken up by TC-Cbl receptor-mediated process by all cells in the body. Lysosomal dissociation of the complex then occurs along with transfer of Cbl to the coenzyme methyl-and Ado-Cbl in the cytoplasm and i...

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Section: Resultsmentioning

confidence: 92%

Crystal structure of human intrinsic factor: Cobalamin complex at 2.6-Å resolution

Mathews¹,

Gordon

Chen³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…Solution competition experiments were therefore performed on the Biacore to verify them as described by other investigators (Cannon et al 2002;Darling and Brault 2004). We exploited the tight affinities of NGF for tanezumab and TrkA as the basis of a blocking assay that used tanezumab in solution to titrate out the binding of NGF to TrkA on the chip (Fig.…”

Section: Kinetics Of Ngf Binding Low-capacity Tanezumab Surfacesmentioning

confidence: 99%

Probing the binding mechanism and affinity of tanezumab, a recombinant humanized anti‐NGF monoclonal antibody, using a repertoire of biosensors

2008

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We describe the use of four complementary biosensors (Biacore 3000, Octet QK, ProteOn XPR36, and KinExA 3000) in characterizing the kinetics of human nerve growth factor (NGF) binding to a humanized NGF-neutralizing monoclonal antibody (tanezumab, formerly known as RN624). Tanezumab is a clinical candidate as a therapy for chronic pain. Our measurements were consistent with the NGF/ tanezumab binding affinity being tighter than 10 pM due to the formation of an extremely stable complex that had an estimated half-life exceeding 100 h, which was beyond the resolution of any of our methods. The system was particularly challenging to study because NGF is an obligate homodimer, and we describe various assay orientations and immobilization methods that were used to minimize avidity in our experiments while keeping NGF in as native a state as possible. We also explored the interactions of NGF with its natural receptors, TrkA and P75, and how tanezumab blocks them. The Biacore blocking assay that we designed was used to quantify the potency of tanezumab and is more precise and reproducible than the currently available cell-based functional assays.

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“…Affinity of the transporting proteins for Cbl still remains a controversial issue with an extraordinary dispersion of the reported equilibrium dissociation constants K d ¼ 10 )9 )10 )15 m [5,7,[10][11][12][13][14][15]. However, the major reasons of this discrepancy are rather artificial.…”

mentioning

confidence: 99%

Application of a fluorescent cobalamin analogue for analysis of the binding kinetics

et al. 2006

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Fluorescent probe rhodamine was appended to 5′ OH‐ribose of cobalamin (Cbl). The prepared conjugate, CBC, bound to the transporting proteins, intrinsic factor (IF) and transcobalamin (TC), responsible for the uptake of Cbl in an organism. Pronounced increase in fluorescence upon CBC attachment facilitated detailed kinetic analysis of Cbl binding. We found that TC had the same affinity for CBC and Cbl (Kd = 5 × 10−15 m), whereas interaction of CBC with the highly specific protein IF was more complex. For instance, CBC behaved normally in the partial reactions CBC + IF30 and CBC + IF20 when binding to the isolated IF fragments (domains). The ligand could also assemble them into a stable complex IF30–CBC–IF20 with higher fluorescent signal. However, dissociation of IF30–CBC–IF20 and IF–CBC was accelerated by factors of 3 and 20, respectively, when compared to the corresponding Cbl complexes. We suggest that the correct domain–domain interactions are the most important factor during recognition and fixation of the ligands by IF. Dissociation of IF–CBC was biphasic, and existence of multiple protein–analogue complexes with normal and partially corrupted structure may explain this behaviour. The most stable component had Kd = 1.5 × 10−13 m, which guarantees the binding of CBC to IF under physiological conditions. The specific intestinal receptor cubilin bound both IF–CBC and IF–Cbl with equal affinity. In conclusion, the fluorescent analogue CBC can be used as a reporting agent in the kinetic studies, moreover, it seems to be applicable for imaging purposes in vivo.

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Equilibrium and Kinetic Analyses of the Interactions between Vitamin B12 Binding Proteins and Cobalamins by Surface Plasmon Resonance

Cited by 30 publications

References 36 publications

Crystal structure of human intrinsic factor: Cobalamin complex at 2.6-Å resolution

Crystal structure of human intrinsic factor: Cobalamin complex at 2.6-Å resolution

Probing the binding mechanism and affinity of tanezumab, a recombinant humanized anti‐NGF monoclonal antibody, using a repertoire of biosensors

Application of a fluorescent cobalamin analogue for analysis of the binding kinetics

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