The metabolism of cobalamin bound to transcobalamin II and to glycoproteins that bind Cbl in HepG2 cells (human hepatoma)

Abstract

The binding, internalization, processing and release of labeled cyanocobalamin (CN[57Co]Cbl) bound to human transcobalamin II (TC II) were studied in HepG2 cells, a line of hepatocytes derived from a human hepatoma. The cells bound the TC II-Cbl by specific, high affinity receptors. Within the cell, the CN-Cbl was promptly freed from TC II and the CN-Cbl converted to more active forms including adenosyl Cbl (AdoCbl) and methyl Cbl (MeCbl). Whereas free labeled Cbl was still present at 72 hours after entry, the cells also bound Cbl to an intracellular binder (ICB) presumed to represent the holo enzymes dependent on Cbl. At levels of TC II that saturated the receptors for TC II-Cbl, much of the Cbl entering the cells remained free and was converted to AdoCbl. Under these circumstances the cells released free Cbl, mostly AdoCbl. Human R type binders of Cbl, which are glycoproteins and some having a terminal galactose, were bound by the HepG2 cells. The binding was characteristic of the receptor system responsive to a terminal galactose, or asialoglycoproteins, but was inconsistent and of low affinity. Cbl bound to R binder was internalized and converted to coenzyme forms of Cbl, but the process was much less effective than when the Cbl entered via the TC II receptor system. It was concluded that the receptors for R-Cbl were unlikely to contribute to the physiologic transport of Cbl in man, but may function in some yet unknown way.

A patient with the inability to maintain in vivo levels of bound cobalamin (Cbl) and manifestations of tissue deficiency of Cbl

“…The TC 11-Cbl of the two subjects, LS and control, were bound equally to the HepG2 cells. Scatchard plots, which were superimposable, gave a K, of 8.6 nM-', the same as observed previously with normal TC I1 [16]. The receptors of L.S.…”

Cyclic activity of the receptors of cobalamin bound to transcobalamin II

“…The HepG2 line was derived from a human hepatoma but retains many properties of the normal hepatocyte (Knowles et al, 1980). The HepG2 cells grow as a monolayer and while division slows at confluency, it does not cease and cells pile up forming multilayers (Knowles et al, 1980;Hall et al, 1985). The diploid human fibroblast represented by the MRC-5 line grows as a monolayer.…”

“…At confluence the growth rate is greatly reduced. All three cells are known to have receptors for the TC 11-Cobalamin (Cbl) complex (TC 11-Cbl) (Youngdahl-Turner et al, 1978;Hall et al, 1985Hall et al, , 1986a.…”

Transcobalamin II Mediated Delivery of Albumin-Bound Hydroxocobalamin to Human Liver Cells

“…The ability of human cells to utilize the OH-[57Co]Cbl bound to HSA was tested in a human liver cell carcinoma (HepG2) in tissue culture ( 15). Cells (5 x lo5) were plated in 35-mm petri dishes for 4 days in 3 ml of Eagle's minimal essential media containing 10% fetal calf serum.…”

“…All dishes contained 1 ml of test sample in minimal essential media without fetal calf serum and were incubated for 6 hr at 37°C. The cells were then processed as described ( 15) and the intracellular radioactivity was determined in a gamma spectrometer. Protein was measured in each cell lysate by the method of Lowry ( 16).…”