Synthesis and secretion of transcobalamin II by cultured astrocytes derived from human brain tissue

Journal of the Neurological Sciences, 122 (1994) 57-60

57

© 1994 Elsevier Science B.V. All rights reserved 0022-510X/94/$07.00 JNS 4211

Synthesis and secretion of transcobalamin II by cultured astrocytes derived from human brain tissue James A. Begley *, Pamela D. Colligan and Richard C. Chu Research and Medical Services (151E), Nutrition Laboratory for Clinical Assessment and Research, Samuel S. Stratton Veterans Affairs Medical Center, 113 Holland Avenue, Albany, N Y 12208, USA

(Received 19 April, 1993) (Revised, received 19 August, 1993) (Accepted 26 August, 1993) Key words: Transcobalamins; Vitamin B12; Cobalamin; Astrocytoma; Brain; Neurobiology

Summary Astrocytes derived from human brain tissue secreted a single cobalamin (vitamin B12, Cbl) binding protein over a 4 day period in culture. Cycloheximide reversibly inhibited the release, and the binding protein was identified as transcobalamin II (TCII) based on molecular size, reaction with anti-human TCII antiserum, precipitation with 2.0 M ammonium sulfate and its ability to bind radioactive cyanocobalamin. It also enhanced the cellular incorporation of the vitamin. Our data show that cultured cells from human brain synthesize and secrete TCII and suggests that at least some of the TCII known to be present in cerebrospinal fluid may originate from within the central nervous system.

Introduction Approximately 25% of the serum cobalamin (vitamin B12, Cbl) is bound to the protein transcobalamin II (TCII) which promotes the cellular incorporation of the vitamin. The TCII-Cbl complex binds to high affinity receptors on various cells and is rapidly internalized into lysosomes where TCII protein is degraded and the Cbl is released into the cyt0plasm..Cobalamin is subsequently converted to methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl) which function as cofactors for the cytoplasmic enzyme 5-methyltetrahydropteroyl-L-glutamate:L-homocysteineS-methyltransferase (EC 2.1.1.13) and the mitochondrial enzyme methylmalonyl-CoA:CoA-carbonylmutase (EC 5.4.99.2) respectively. Various human tissues and cells have been shown to synthesize TCII including bone marrow (Frater-Schroder et al. 1985), hepatocytes (Hall et al. 1985), fibroblasts (Berliner and Rosenberg 1981), and umbilical vein endothelial ceils (Quadros et al. 1988). Cobalamin is essential for the function of the human central nervous system (CNS), and tissue depletion of this vitamin leads to distinct neurologic disorders (Green and Jacobsen 1990; Hall 1990).

* Corresponding author. Tel.: (518) 462-3311, ext. 2394; Fax: (518)472-7019. SSDI 0022-510X(93)E0222-U

Little is known about Cbl transport and metabolism in the human CNS. Human cerebrospinal fluid (CSF) contains both R-binder and TCII with the latter predominating (Finkler et al. 1970; Lazar and Carmel 1981; Hansen et al. 1985). R-binder is a Cbl binding protein which is immunologically different from TCII. Athough it carries approximately 75% of the Cbl in serum, a definitive function has never been established. CSF contains 5-108 p g / m l of Cbl (Frenkel et al. 1971; Hansen et al. 1985) which is bound predominantly to TCII (Lazar and Carmel 1981) and may be mostly in the form of AdoCbl (Linnell et al. 1974). Lazar et al. (1981) showed that TCII in CSF enhanced the uptake of Cbl by neonatal and adult human brain homogenates. Although CSF contains TCII and TCII-Cbl, it is not known whether the cells of the CNS are capable of synthesizing the protein a n d / o r whether it is transported from plasma through the blood-brain barrier or blood CSF barrier. It has been suggested that TCII is synthesized by cells of the CNS since the C S F / p l a s m a ratio of TCII is higher when compared to other plasma proteins (Hansen et al. 1985; Hansen and Nexo 1987). Recently, Pezacka et al. (1992) showed that human glial cells in culture were capable of incorporating and metabolizing TCII-Cbl to coenzyme forms. In addition, they showed that an antibody to TCII inhibited the uptake of free Cbl. They postulated that this effect was due to the binding of the antibody to the

TCII secreted by the cells and inhibition of uptake of the TCII-Cbl complex. The present study directly confirms the preliminary findings of Pezacka et al. (1992) and shows that cells cultured from human brain tissue are capable of synthesizing a Cbl binding protein which we characterize as TCII.

Materials and methods

Cells and culture conditions The human astrocytoma cell line STT was obtained from the American Type Culture Collection (ATCC No. CRL 1718). To study TCII synthesis, confluent cells (approximately 1.7 × 106) were cultured for 96 h with 3 ml of Cbl free RPMI medium containing 1 mM L-glutamine and 10% human serum from which both R-binder and TCI1 were removed by immunoadsorption (Begley 1983). Two sets of duplicate flasks contained 5 / z g / m l of cycloheximide and a third set contained none. One ml of medium was removed from each flask at 24, 48 and 72 h, frozen for analysis and each flask replenished with 1 ml of the appropriate medium. At 24 h, the medium from one set containing cycloheximide was completely replaced with fresh medium without cycloheximide to allow the cells to recover from the effects of the inhibitor. At the end of the experiment (96 h), the total medium was frozen in aliquots and the cells were stained with trypan blue and counted for cell number and viability. Analysis of Cbl binding proteins in culture medium The Cbl binding proteins released into the culture medium at each time interval were analyzed for radioactive cyanocobalamin (CN[57Co]Cbl) binding ability using albumin-coated charcoal as described (Green and Hall 1980). In order to determine what Cbl binder was released into the medium, the albumin-charcoal supernatant from the 96 h time point was further analyzed by Sephadex G-200 gel chromatography as described (Begley 1983). Immunological identification of the astrocytic Cbl binder as TCII A portion of the 72 h conditioned medium was precipitated with 2.0 M (NH4)2SO 4 (Begley 1983). The precipitate was dissolved with 2 ml of DPBS, labelled with CN[57Co]Cbl for 30 min at 37°C and analyzed by Sephadex G-200 chromatography. The protein bound CN[S7Co]Cbl was concentrated in a Diaflow Ultrafilter fitted with a PM-10 membrane (Amicon Corp.), reacted with rabbit antibody to human TCII and rechromatographed by gel filtration. Reaction of the antibody with TCII is detected as a shift in elution of the TCII-CN[SVCo]Cbl to the excluded volume of the column (Finkler et al. 1970; Begley 1983).

Determination of the biological actmi(v of the Cb/ binding protein released by astrocytes One ml of the medium from the 96 h time point was labelled with excess CN[57Co]Cbl for 30 rain at 37°C and dialyzed overnight against 100 volumes of Eagle s minimal essential medium. In addition, saliva and Cohn fraction were also processed as above and used as sources of R-CN[57Co]Cbl and TClI-CN[57Co]Cbl respectively. Three day cultures of human fibroblast cells, MRC-5, were cultured as described (Hall et al. 1987) and exposed to 48 p g / m l of free CN[57Co]Cbl or CN[57Co]Cbl bound to either R-binder, TCII or the binder present in the 96 h conditioned medium from the astrocytes. After 24 h, the cells were harvested as described (Hall et al. 1987) and the internalized CN[SVCo]Cbl quantified in a gamma spectrometer.

Results

Fig. 1 shows the cumulative secretion of Cbl binding proteins into the culture medium conditioned by STT astrocytoma cells over a 96 h interval. There was a linear increase in the amount of binder released which was completely inhibited by the presence of 5 u g / m l cycloheximide. At the end of the study (96 h) cells in the absence of cycloheximide synthesized 86 pg of T C I I / 1 0 6 cells. Removal of the inhibitor at 24 h enabled the ceils to resume synthesis of TCII to within 60% of that secreted in the absence of cycloheximide by 96 h. At the termination of the study, all cells were > 95% viable as determined by trypan blue exclusion. Analysis of the albumin-charcoal supernatant from the 96 h time point of Fig. 1 by Sephadex gel filtration showed that only one Cbl binding protein was secreted

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Days in Culture Fig. 1. Cummulative synthesis of Cbl binding proteins by S ] T astrocytoma cells over a 4 day interval. Cells were cultured either without cycloheximide (o . . . . . . e) or in the presence of 5 / z g / m l (o o). At 24 h, the medium from one set of duplicate flasks

containing cycloheximidewas replaced with medium without the inhibitor ( • • ) to study the recoveryof Chl binding protein synthesis. Each point represents the average of duplicate cultures.

59 TABLE 1 INCORPORATION OF ASTROCYTIC TCII INTO MRC-5 FIBROBLASTS Values represent duplicate cultures for each parameter. Sample analyzed TCII in 96 h culture medium from astrocytoma cells labelled with CN[57Co]Cbl human TCII-CN[STCo]Cbl human R-CN[57Co]Cbl free CN[57Co]Cbl

pg CN[57Co]fbl internalized/106 cells

5.2, 6.1, 0.1, 0.6,

5.7 5.8 0.2 0.7

and it eluted from the calibrated column where human serum TCII elutes (data not shown). In order to determine the immunological nature of the binder secreted, the Cbl binding proteins in 72 h culture medium were precipitated with 2.0 M (NH4)2504, labelled with CN[57Co]Cbl and analyzed by Sephadex G-200 gel filtration. Greater than 95% of the protein-bound CN[57Co]Cbl eluted from the column similar to human TCII. Reaction of this proteinbound CN[57Co]Cbl peak with anti-TCII antibody followed by rechromatography resulted in shifting of the radioactivity to the excluded volume of the column. This data shows that the Cbl binding protein in the culture medium was TCII since it precipitated with 2.0 M (NH4)2SO 4 (Begley 1983), had a molecular size identical to human serum TCII as determined by gel filtration (Begley 1983), and reacted with antibody specific for human TCII (Finkler et al. 1970). Table 1 shows that the Cbl binder secreted into the culture medium performed the biological function of TCII by facilitating the incorporation of Cbl into cells similar to TCII isolated from human serum. Free CN[57Co]Cbl or R-CN[57Co]Cbl did not enter the cells to any significant degree.

reductase (EC 1.6.99.8). The mutase was not significantly affected. Similar decreases in Cbl deficient fibroblasts were not observed over the 6 weeks of the study, indicating that cells of the nervous system might be more sensitive to Cbl deficiency. They also suggested that these glial cells were synthesizing TCII since culturing them in the presence of anti-TCII antibody inhibited the uptake of free CN-Cbl. Since Cbl is not synthesized in human cells, it must enter the brain and CSF from the blood. How this Cbl gains entry into the CNS and what role TCII plays in its transport to and within the brain is unknown. Several theories have been proposed to explain the relative enrichment of a particular protein, such as TCII, in the CSF: (1) selective degradation of some plasma proteins during transport through the choroid plexus, (2) a discriminating mechanism for the transcellular transfer of plasma proteins from blood to brain, (3) a functional leak in the blood-brain barrier, and (4) synthesis of the protein by the brain (Tu et al. 1991). Our results directly confirm that cells derived from brain tissue synthesize TCII and this may account at least in part for the levels of TCII present in CSF. It is also possible that cells of the brain capillary endothelium a n d / o r the choroid plexus secrete TCII into the brain fluids. Much needs to be answered about how Cbl enters the CNS, what cells within the brain require Cbl and how do they obtain it, and what the role of TCII is in these processes. Transcobalamin II could function in Cbl recycling and transport within the brain or could serve as a binding protein for free Cbl entering the fluids of the CNS from the choroid plexus or brain capillary endothelial cells. The human choroid plexus and brain capillary endothelial cells may have receptors for plasma TCII-Cbl which facilitate incorporation of Cbl through the cell and into the CSF or interstitial fluid as an intact TCII-Cbl complex or as free Cbl. Acknowledgements The authors would like to thank Dr. Arnulf H.

Discussion

Anatomical lesions of the CNS can occur in either acquired Cbl deficiency (Green and Jacobsen 1990) or in genetic defects of Cbl metabolism (Hall 1990). Bhatt and Linnell (1990) studied Cbl influx into the brain of the rat in vivo and suggested that a carrier mediated process was involved. Pezacka et al. (1992) were the first group to study Cbl metabolism in cultured human brain cells. They showed that astrocytes incorporated either free CN-Cbl or CN-Cbl bound to rabbit TCII and converted Cbl to MeCbl and AdoCbl. Growth of the cells in Cbl-deficient medium resulted in an impairment in the formation of AdoCbl and MeCbl as well as decreased activity of methyltransferase and Cbl

Koeppen for his helpful discussions during the preparation of the manuscript. This work was supported by the Research Service of the Department of Veterans Affairs.

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