Kidney International, Vol. 39 (1991), pp. 289-294

Studies on the transcobalamin receptor in hog kidney

1 SHOJI YAMADA , LEENA RIITTINEN, RAIMO MAJURI, MORIMICHI FUKUDA, and RALPH GRASBECK

Minerva Foundation Institute for Medical Research, Helsinki, Finland, and Department of Laboratory Diagnosis, Sapporo Medical College, Sapporo, Japan

Studies on the transcobalamin receptor in hog kidney. The binding of Cbl-TC complex was reabsorbed and degraded by a similar the cobalamin-trans cobalamin complex by its solubilized receptor from process as low-molecular-weight in general. The cel­ hog kidney membrane was studied. The receptor bound the complex in a system containing bivalent cations, and the affinity was dependent on lular uptake of the Cbl-TC complex begins with binding of the the NaCI concentration but not on temperature. The binding of coba­ complex to a specific surface receptor [13, 14]. This report lamin-transcobalamin to the receptor had an association constant of describes some characteristics of the Cbl-TC receptor solubi­ approximately 4.6 x 109 liter/mol and it was saturable and highly lized from hog kidney. specific as competition by other proteins was not observed. The receptor had higher affinity for the cobalamin-transcobalamin complex Methods (holo-TC) than for transcobalamin (apo-TC). Basic amino compounds known to interfere with tubular reabsorption of proteins did not inhibit Reagents the binding. Studies on subcellular fractions supported the view that the receptor was located on the brush border membrane of the kidney.2 All reagents were commercially available guaranteed-grade chemicals. Hog serum (Fraction V, Sigma Chemical Co., St. Louis, Missouri, USA) was passed through a Cbl­ Sepharose column before use to remove Cbl binders which Transcobalamin (TC, formerly designated as transcobalamin eluted like TC in gel filtration. 57Co-Cbl with a specific activity II) is a cobalamin (Cbl)-binding polypeptide with a molecular of 200 Ci/g was from the Radiochemical Centre (England, UK). weight of 38,000 to 43,000 daltons in humans [1, 2]. Under Hog [15] and [16] were purified as physiological conditions it accounts for at least 80% of the described before. approximately 0.75 nmol/liter of the unsaturated Cbl-binding capacity of plasma [1], and it facilitates the uptake of Cbl by a Preparation of receptor extract from kidney with detergent variety of cells both in vitro [1, 3, 4] and in vivo [5]. Fresh hog kidneys from a slaughterhouse were stored at Low-molecular-weight proteins are generally filtered into the -20°C until used. Receptor extract was prepared from the primary urine, from which they are efficiently reabsorbed by cortex of a kidney by procedures similar to those described for proximal tubule cells. They are then degraded in lysosomes and preparation of IF-receptor from ileal mucosa [17]. The unsatu­ the degradation products return to the circulation [~]. rated Cbl-binding capacity of the extract was usually less than Radioactive Cbl, administered either subcutaneously or 0.4 pmolliiter as determined by the coated charcoal method orally to rats, first accumulates mainly in the kidneys and then [18]. it migrates to other organs [9-11]. Since almost all of the administered Cbl binds to TC in blood except when an ex­ Partial purification of TC from hog serum tremely large dose is injected, the strong accumulation in the This was performed essentially as described [19]. kidney suggests a high capacity of this organ to take up TC-bound Cbl. Newmark [12] reported that both microbiolog­ Conversion of holo receptor into apo receptor and partial ical Cbl activity and intramuscularly injected 57Co-cyanocobal­ purification of receptor amin was concentrated more in the lysosomal fraction of kidney A part of the TC receptor in the receptor extract was than in other subcellular fractions, and speculated that the suspected to be saturated with either TC or Cbl-TC (holo receptor). To convert the holo receptor into the apo form and to remove contaminating proteins, the receptor extract was proc­ 1 Present address: Department of Home Economics, Hokkaido Uni­ essed as follows: Na -EDTA (Titriplex III, Merck, Germany) versity of Education, Sapporo, Japan. 2 2 Abbreviations are: CN-Cbl, cyanocoblamin; Cbl, cobalalTlin or was added to a kidney extract to a final concentration of 25 mmolliiter and incubated at room temperature for 30 minutes. vitamin B12 ; 57Co_Cbl, 57Co-Iabeled cyanocobalamin; IF, intrinsic fac­ tor; HC, (R-binder); TC, transcobalamin; Vo, void volume; The mixture was filtered through a Sephadex G-200 column pp, partially purified. equilibrated with EDTA-receptor buffer (vide infra). Fractions Received for publication January 21, 1988 near the void volume (Vo), which contained Blue Dextran 2,000 and in revised form October 5, 1990 in a concentration more than one-tenth of that in the fraction of Accepted for publication October 10, 1990 Yo, were pooled, concentrated by ultrafiltration, dialyzed © 1991 by the International Society of Nephrology against three changes of extracting buffer for 72 hours and for 48

289 290 Yamada et al: Transcobalamin receptor

Receptor activity assayed on a kidney extract with these two columns differed less than 4%. To obtain a saturation curve for binding of Cbl-TC to receptor, gel filtrations were made using a (41021) TSK G 3,OQO SW column. The running buffers for all the three L columns were 50 mmolliiter Tris-HCI, pH 7.4, containing 0.05% 1 , Triton X-100, 0.15 mol/liter NaCl, 1.5 mmol/liter NaN3 and 25 either 1 mmol/liter CaCl2 (receptor buffer) or 10 mmol/liter Na2-EDTA (EDTA-receptor buffer).

Protein estimation was determined by the original [20] or modified Lowry method [21] using bovine as a standard. In some pp TC preparations also absorbance at 280 nm was used.

Purification of IF and HC Hog gastric mucosal scrapings were homogenized. Ammoc nium sulfate was added to a concentration of 30% saturation, the precipitate was collected by centrifugation and the salt concentration was increased to 65% saturation. The resulting precipitate containing HC was dissolved and after addition of cobinamide it was applied on a Cbl-Sepharose column [22]. The breakthrough fraction which contained most of the cobina­ mide-HC complex was dialyzed against 7.5 mol/liter guanidine­ 50 100 HCI to remove cobinamide and then against phosphate buffer to Fraction number remove guanidine. The dialyzed preparation was applied again on Cbl-Sepharose, and after thorough washing HC was eluted Fig. 1. Assay of TC receptor by gel filtration. Partially purified (pp) receptor was incubated with pp TC and 57Co_Cbl for 60 min at room with guanidine. temperature. The mixture was filtered through a Sephadex G-200 Hog IF was purified from the supernatant of the 65% ammo­ column (2.5 x 50 cm) equilibrated with receptor buffer. Fractions of nium sulfate step described above. To the supernatant was 3.34 ml were collected and counted for 57CO. Symbols are: (circles) added cobin amide to block HC and the mixture was applied complete assay system; (triangles) control system lacking receptor onto a Cbl-Sepharose column. After thorough washing, IF was preparation; (squares) control system lacking pp TC. eluted with guanidine, filtered through a TSK G 3,000 SW column and fractions corresponding to the elution volume of IF were collected. hours against extracting buffer containing CaCl2 in a concentra­ tion of 10 mmollliter with a change of buffer at 24 hours. In the following this preparation is called partially purified (pp) recep­ Removal of Cbl from holo-TC tor. Cbl contained in the pp TC preparation was removed by dialyzing the preparation against 10 volumes of 5.0 mol/liter Assay of receptor activity guanidine (120 hr) and then against 100 volumes of 50 mmol/liter Pp TC with a Cbl-binding capacity of 0.25 pmol was incu­ sodium phosphate buffer, pH 6.0, containing 0.2 mol/liter NaCl bated with 0.32 pmol of 57Co-Cbl for 30 minutes. The solution for 120 hours each with four dialysate changes. The Cbl was mixed with an appropriate volume of pp receptor, brought concentration in the preparation was estimated with a "Phade­ up to 1.0 ml with receptor buffer (vide infra), incubated at room bas B12 test" (Pharmacia, Uppsala, Sweden). temperature for 60 minutes on an end-to-end mixer and then filtered through a Sephadex G-200 column. A representative Assay of enzyme activities elution profile is shoWll in Figure 1. Radioactivity of fractions near Vo (hatched area in Fig. 1) was summed up and used as the Alkaline phosphatase was assayed by a modified method of criteria for receptor activity. Controls lacking either the recep­ Kind and King [23] using phenyl phosphate as substrate. tor preparation or TC were run separately through the same Leucine aminopeptidase was assayed by a modified method of column. Goldbarg and Rutenburg [24] using L-Ieucyl-p-diethyl-amino­ anilide as substrate. y-Glutamyltranspeptidase was assayed by Gel filtrations a modified method of Orlowski and Meister [25] using y-glu­ They were mostly performed in a Sephadex G-2oo column tamyl-p-N-ethyl-N-hydroxyethyl aminoanilide as substrate. (2.5 X 50 cm, 4°C). To investigate the effect of NaCI concen­ Cytochrome C oxidase was assayed by the method of Wharton tration on the formation of the Cbl-TC-receptor complex, a and Tzagoroff [26]. Glucose-6-phosphatase was assayed by the smaller column of 0.9 x 30 cm was used. The flow rates were method of Hiibscher and West [27], and J3-g1ucuronidase by the 13.3 mllhr for the bigger and 1.9 mllhr for the smaller column. method of Plaice [28]. Yamada et al: Transcobalamin receptor 291

100 x _a.Q) 250 o E +-' 0 :Jc: ....'" o 0 '0 E+-' \ .E co ~ 200 Q) '" !~ ,; .2: ~ > ~u t(2) c: a;~ 50 a:...!. -0 .D ~ u (2) Q) > 150 0 '"~ 1i u 6 ....u III 0 0 45 60 90 120 50 Incubation period, minutes Fig. 3. Rate of formation of Cbl-TC-receptor complex. Pp receptor was incubated with pp TC and 57Co-Cbl for a period indicated on the abscissa. Formation of Cbl-TC-receptor complex at each incubation o time was assayed as usual. Mean values and 1 so of 2 to 5 determina­ 0.3 0.6 0.1 0.15 tions are depicted by closed circles and vertical bars, respectively. Concentration of NaCI, mol/liter Figures in parentheses denote number of determinations. *Differences of values at 0 and 15 min is statistically insignificant; 0.10 < P < 0.20. Fig. 2. Injiuence of NaCI concentration on Cbl-TC-receptor complex formation. Pp receptor was incubated with pp TC and 57Co-Cbl for 60 min at room temperature at different concentrations of NaCI. The mixtures were filtered through a Sephadex G-200 column equilibrated activity in both control system (pp TC + 57Co-Cbl and receptor with running buffer containing the same concentration of NaCI as the preparation + 57Co_Cbl). incubation mixture. Symbols are: (solid column) receptor + TC + 57Co-Cbl; (hatched column) TC + 57Co-Cbl ; (open column) receptor + Ten mmol/liter Na2-EDTA reduced the formation of the 57Co-Cbl. Cbl-TC-receptor complex by more than 98%. EDTA treatment was used to convert holo to apo receptor as described in Methods. The amount of apo receptor in the pp receptor preparation so obtained increased, on the average of five Results experiments, by 4.4 times that of original extract. Assay of receptor for Cb/-TC Effect of temperature on binding A suitable ionic strength for the gel filtration buffer was found Lowering incubation temperature from room temperature to be important because TC aggregated at low ionic strength (22°C) to 4°C decreased binding ofCbl-TC to receptor to 83.6 ± [29, 30] and, on the other hand , high ionic strength inhibited 7.8% in four experiments. The difference was, however, statis­ protein-protein binding, including binding to the receptor. Ac­ tically insignificant. cordingly, we first investigated the effects of N aCI concentra­ tion on the receptor assay. Rate of binding At low concentration of NaCI, such as 0.0375 and 0.075 As shown in Figure 3 binding of Cbl-TC to receptor increased mol/liter, a large quantity of 57Co-Cbl emerged near the Vo in to reach a maximum followed by a gradual decrease with the complete assay system (receptor preparation + pp TC + prolonged incubation time. In some occasions binding at time 0 57Co-Cbl). However, most of that radioactivity is probablY due was unexpectedly high, although the variation between exper­ to aggregated TC because a control (pp TC + 57Co-Cbl) gave a iments was quite large. Receptor assay was carried out by gel comparable quantity of 57Co-Cbl in the same region. filtration which takes several hours, and reaction of Cbl-TC In the control system the amount of 57Co-Cbl near Vo with its receptor is imagined to continue at least for some time became insignificant as the concentration of NaCI was in­ after the onset of gel filtration. Incubation time indicated on the creased to 0.15 molfliter (Fig. 2). At NaCl concentrations above abscissa of Figure 3 is in this sense inevitably inaccurate. 0.3 molliiter the 57Co-Cbl near the Vo diminished markedly, suggesting inhibition of formation of Cbl-TC-receptor complex. Saturability of receptor The recovery of 57Co-Cbl in the effluent was more than 90% Binding of 57Co-Cbl-TC to receptor seemed saturable (Fig. when the NaCI concentration was above 0.075 mol/liter. 4) . By Scatchard analysis the association constant was calcu­ In the following experiments assay of receptor was per­ lated to be 4.6 x 109 liter/mol. When a tenfold excess of formed in a buffer containing 0.t5 molliiter NaCI. The amount non-radioactive Cbl-TC was added the binding of 57Co-Cbl was of Cbl-TC receptor was calculated from the 57Co-Cbl found reduced by 83% (Table 1). This also suggested saturability of near the Vo in the complete assay system minus the analogous binding of Cbl-TC to receptor and specificity of the assay 292 Yamada et al: Transcobalamin receptor

Table 2. Reversibility of binding of Cbl-TC to its receptor 15 Amount of 57Co_Cbl relative to that of Condition of storage of 57Co-Cbl-TC-receptor which was applied on Cbl-TC-receptor before re-filtration, % re-filtration (2nd Cbl-TC-receptor Cbl-TC Free Cbl filtration) region region region

~~~ 4°C, 0 hr 42" (40,43)b 0(0,0) 2 (0,4) 4°C, 112 hrs 32 (30,34) 0(0,0) 0(0,0) 2ZOC, 112 hrs 27 (32,22) 0(0,0) 8 (6,10) 4°C, 16 hrs, with added 0(0,0) 106 (102,110) 19 (20,17) EDTA

a Values are means of 2 experiments b Figures in parentheses are values of each two determinations

brated with receptor buffer (second gel filtration). About 40% or less of the Cbl-TC-receptor was recovered as such (Table 2). The low recovery is probably due to absorption of TC or Concentration of 57Co-Cbl-TC, pmol/liter receptor moiety of Cbl-TC-receptor complex to gel matrix and not to degradation of the complex. This is indicated by the Fig. 4. Saturability o/binding o/Cbl-TC to receptor. A fixed amount of pp receptor was incubated with 57Co-Cbl-TC at concentration indicated absence of any other peaks in the second gel filtration. on the abscissa and receptor-bound 57Co-Cbl-TC assayed as described Increasing time and temperature of storage of Cbl-TC-recep­ in the text. Inset: similar data treated according to Scatchard. Inset tor between the first and second gel filtration did not markedly abscissa: concentration of bound ligand (pmollliter). Inset ordinate: reduce the recovery of Cbl-TC-receptor from the second gel ratio of concentration of bound ligand to that of free ligand. filtration. Thus binding of Cbl-TC and the receptor seems rather stable. Table 1. Competition for the receptor with Cbl-hog TC by some proteins and basic amino compounds Subcellular localization of receptors 57Co-Cbl-TC bound The microvillous fraction was prepared from homogenate of to receptor kidney cortex [31]. Receptor extracts were prepared from both Molar whole homogenate and the microvillous fraction. Change of ratio to % o/control 57CO_ Number of Extreme specific activity of receptor for TC (Cbl bound via TC/mg Test substance Cbl-TC experiments Mean values protein) was compared with some of marker enzymes for subcellular organeles. As shown in Table 3, the ratio of specific Hog serum albumin 1,000 2 93.8 92.1-95.5 Hog transferrin 1,300 2 102.2 93.0-11 1.3 activity of receptor in microvillous fraction to that in whole Hog hemopexin 1,000 2 92.9 84.5-101.3 homogenate was roughly same as the corresponding ratio of 1,000 2 71.9 61.0-81.4 marker enzymes for brush border membranes, that is, alkaline L-Lysine 100,000 2 121.6 115.5-127.7 phosphatase, leucine aminopeptidase and y-glutamyl-transpep­ L-Arginine 10,000 2 89.7 86.0-93.3 6-Amino caproic 100,000 2 112.6 9\.6-133.5 tidase. It was quite different from those of cytochrome C acid oxidase, which is a marker for mitochondria, glucose-6-phos­ Cbl-hog HC 29 2 87.4 85.2-89.6 phatase for microsome and J3-glucuronidase for lysosome frac­ Cbl-hog IF 6 1 70.8" tion. These findings seem to suggest that the receptor is mainly Partially purified 10 5 33.3 14.7-7\.6 hogTC localized in the brush border membrane. Partially purified 10 4 17.3 13.7-23.1 Cbl-hog TC Specificity of binding of Cbl-TC to receptor None of the proteins and basic amino compounds that we a Single experiment due to lack of the material tested clearly interfered with the binding of Cbl-TC to the receptor, the only exception being Cbl-HC (Table 1). In prelim­ inary experiments, addition of small amounts of Cbl-hog IF also system. Addition of similar amounts of pp TC, from which most caused a somewhat larger decrease of binding of 57Co-Cbl-TC of the endogenous Cbl had been removed as described above than addition of other proteins. (apo TC), -in general caused somewhat less reduction of binding of 57Co-Cbl to the receptor, with a mean of 67%. However, the Discussion difference between the effects of holo and apo TC was not The glomerular sieving coefficient for TC and its Cbl complex statistically significant (Table 1). can be estimated to be about 10% based on their molecular weight and electric charge. To prevent spillover of significant Reversibility of binding amounts of TC into urine it seems necessary that the tubular 57Co-Cbl-TC-receptor complex was obtained by gel filtration cells reabsorb it. similar to assay procedure for the receptor (first gel filtration). It Lindemans, van Kapel and Abels [32] reported on the uptake was again filtered through a Sephadex G-200 column equili- of Cbl-TC by isolated tubular cells of rats. According to the Yamada et al: Transcobalamin receptor 293

Table 3. Specific activities of receptor for Cbl-TC and marker enzymes in whole homogenate and microvillous fractions from frozen hog kidney cortex

Specific activity Relative specific Whole activity Known homogenate Microvillous (enrichment) localization (H) fraction (MV) MV/H Receptor for TC 2.22 ± 0.65" 16.96 ± 5.73" 7.64 Alkaline phosphatase Brush border 149 ± 31 b 825 ± 30b 5.54 membrane Leucine aminopeptidase Brush border 184 ± lOe 1.400 ± 7e 7.61 membrane y-Glutamyltranspeptidase Brush border 0.25 ± 0.04d 1.33 ± 0.20d 5.29 membrane Cytochrome C oxidase Mitochondrion 4.52 ± 0.87e 0.90 ± om e 0.20 Glucose-6-phosphatase Microsome 4.39 ± 0.94f 3.87 ± 0.76f 0.88 (3-Glucuronidase Lysosome 0.203 ± 0.056g 0.202 ± 0.027g 0.99 Values are means ± 1 so of three experiments. " fmollmg protein b K-A U/mg protein e mU/mg protein d IU/mg protein e k/mg protein f f.Lg P/mg protein g U/mg protein

report, the uptake was depressed by metabolic inhibitors, was human fibroblast [41], liver [40], placenta [37, 38] and Ehrlich less saturable, decreased dose-dependently by addition of large as cite cells [3], and is probably necessary for the uptake of such amounts of other proteins and marginally by lysine. With the Cbl that the organ itself consumes or stores it. The other, exception of the last item, these characteristics are common studied by Lindemans et al [32], is presumably involved in features in the reabsorption of low-molecular-weight proteins, tubular reabsorption. that is, the system is inhibited by basic amino compounds such Thus it seems that the kidneys play a unique role in Cbl as lysine, arginine, ornithine and 6-amino caproic acid [33], and metabolism in some animal species: They store Cbl when it is is characterized by low-affinity and low-selectivity [34], but supplied over body demand and slowly leak it to other organs. high transport capacity [6, 35]. The kidneys seem to catch and prevent the loss of Cbl by two However, the present results indicate that the kind of uptake mechanisms, unspecific reabsorption of Cbl-TC and a high of Cbl-TC by the kidney that we studied differs from the usual capacity-specific system for the uptake of Cbl-TC, probablY by tubular reabsorption of low-molecular-weight proteins. The non-tubular cells. binding of Cbl-TC to the receptor was not inhibited by basic amino compounds, it was highly selective and clearly saturable. Acknowledgments Moreover, in this study the receptor had somewhat higher This work was supported by grants from the Sigrid Juselius Founda­ affinity for holo-TC than for apo-TC. Because most of the TC in tion, The Finnish-Norwegian Medical Foundation and the Nordisk plasma is in the apo-form, it may be taken up by a different Insulinfond. Shoji Yamada gratefully acknowledges a travel grant from system such as tubular reabsorption. the Academy of Finland. We thank Dr. Ilkka Kouvonen and Virve Scott, Bowman and Cookley [36] reported that the binding of Wahlstedt, M.Sc., for helpful suggestions and Mrs. Seija Nordberg for technical assistance. 57Co-Cbl-TC to the plasma membrane of rat kidney was satu­ rable and effectively inhibited by unlabeled Cbl-TC, and some­ Reprint requests to Ralph Griisbeck, M.D., Minerva Foundation what less strongly by apo-TC. This is compatible with our Institute for Medical Research, Tukholmankatu 2, SF-00250 Helsinki, findings. Our ~)(udies and those of Scott et al [36] were made on Finland. the binding of Cbl-TC to a preparation from whole cortex of kidney. References The association constant for the Cbl-TC-receptor system 1. ALLEN RH: The plasma transport of vitamin BI2• Brit J Haematol agreed closely with those reported for the binding of human 33:161-171,1976 Cb!-TC to a membrane preparation of placenta [37-39] and of 2. NATORI H: Physicochemical analyses of vitamin BI2 binding pro­ rat Cbl-TC to liver plasma membrane [40]. The binding to the teins in sera, gastric juice, and leucocytes. Tumor Res 6:1-79, 1971 3. PARANCHYCH W, COOPER BA: Factors influencing the uptake of receptor was inhibited by NaCl in a concentration-dependent cyanocobalamin (vitamin BI2) by Ehrlich ascites carcinoma cells. manner, suggesting that the interaction is more likely ionic than Biochim Biophys Acta 60:393-403, 1962 hydrophobic. Similar observations were reported by Nex~ and 4. FINKLER AE, HALL CA: Nature of the relationship between Hollenberg [37] on human placenta and rabbit liver. vitamin BI2 binding and cell uptake. Arch Biochem Biophys 120: 79-85, 1967 Hence, the conclusion seems justified that the kidney con­ 5. SCHNEIDER RJ, BURGER RL, MEHLMAN CS, ALLEN RH: The role tains two systems that bind Cbl-TC. One of them studied by us and fate of rabbit and human trans cobalamin II in the plasma and Scott et al [36] has properties similar to the TC receptors in transport of vitamin BI2 in the rabbit. J Clin Invest 57:27-38, 1976 294 Yamada et al: Transcobalamin receptor

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Clin Sci 67:299- and patients with cancer and other diseases. Cancer 1l:283-291, 306, 1984 1958 43. OKUDA K: Relationship between intake of vitamin B12 and its 25. ORLOWSKI M, MEISTER A: '}'-Glutamyl-p-nitroanilide: A new con- storage by the kidney in the rat. J Nutr 77: 131-136, 1962