Investigation of Receptor-Mediated Cyanocobalamin (Vitamin B12

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Investigation of receptor-mediated cyanocobalamin (vitamin B12) transport across the inner blood-retinal barrier using fluorescence-labeled cyanocobalamin Yuri Kinoshita, Kagayaki Nogami, Ryuta Jomura, Shin-ichi Akanuma, Hajime Abe, Masahiko Inouye, Yoshiyuki Kubo, and Ken-ichi Hosoya Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00617 • Publication Date (Web): 02 Jul 2018 Downloaded from http://pubs.acs.org on July 4, 2018

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Molecular Pharmaceutics

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Investigation of receptor-mediated cyanocobalamin (vitamin B12) transport across the

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inner blood-retinal barrier using fluorescence-labeled cyanocobalamin

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Yuri Kinoshita1, Kagayaki Nogami2, Ryuta Jomura1, Shin-ichi Akanuma1, Hajime Abe2,

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Masahiko Inouye2, Yoshiyuki Kubo1*, and Ken-ichi Hosoya1

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1

8

University of Toyama, Toyama, Japan

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2

10

Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences,

Department of Chemical Biology, Graduate School of Medicine and Pharmaceutical Sciences,

University of Toyama, Toyama, Japan

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Running title: Cyanocobalamin transport at the inner blood-retina barrier

13 14

Kinoshita Y, Nogami K and Kubo Y made contribution to the present work equally.

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*Author for correspondence: Yoshiyuki Kubo, Ph.D.

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University of Toyama, Graduate School of Medicine and Pharmaceutical Sciences, Department

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of Pharmaceutics

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Address; Sugitani 2630, Toyama, 930-0194, Japan

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E-mail: [email protected]

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Voice: +81-76-434-7507;

FAX: +81-76-434-5172

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract

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The blood-to-retina supply of cyanocobalamin (vitamin B12) across the blood-retinal

4

barrier (BRB) was investigated by synthesizing a fluorescence-labeled cyanocobalamin

5

(Cy5-cyanocobalamin). In the in vivo analysis following internal jugular injection of

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Cy5-cyanocobalamin, confocal microscopy showed the distribution of Cy5-cyanocobalamin in

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the inner plexiform layer (IPL), the outer plexiform layer (OPL) and the retinal pigment

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epithelium (RPE). In the in vitro analysis with TR-iBRB2 cells, an in vitro model cell line of the

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inner BRB, Cy5-cyanocobalamin uptake by TR-iBRB2 cells exhibited a time-dependent

10

increase after preincubation with transcobalamin II (TCII) protein, during its residual uptake

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without preincubation with TCII protein. The Cy5-cyanocobalamin uptake by TR-iBRB2 cells

12

was significantly reduced in the presence of unlabeled cyanocobalamin, chlorpromazine and

13

chloroquine, and was also significantly reduced under Ca2+-free conditions. Confocal

14

microscopy of the TR-iBRB2 cells showed fluorescence signals of Cy5-cyanocobalamin and

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GFP-TCII protein, and these signals merged with each other. The RT-PCR, Western blot and

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immunohistochemistry clearly suggested the expression of TCII receptor (TCII-R) in the inner

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and outer BRB. These results suggested the involvement of receptor-mediated endocytosis in

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the blood-to-retina transport of cyanocobalamin at the inner BRB with implying its possible

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involvement at the outer BRB.

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Keywords: cyanocobalamin; vitamin B12; blood-retinal barrier; transcobalamin II receptor;

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receptor-mediated transport

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1. Introduction

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Vitamin B12, which includes cyanocobalamin, hydroxocobalamin, methylcobalamin and

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adenosylcobalamin, is a water-soluble nutrient possessing a cobalt and corrin ring structure. In

5

mammals, the major source of vitamin B12 is hydroxocobalamin and cyanocobalamin from the

6

diet and commercially available supplements, respectively, and, in particular, cyanocobalamin

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is superior to other vitamin B12 molecules in terms of temperature and alkaline stability,

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suggesting its wide clinical application.1 Hydroxocobalamin and cyanocobalamin are

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enzymatically converted to the biologically active forms of vitamin B12, methylcobalamin and

10

adenosylcobalamin,

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(5-methyltetrahydrofolate-homocysteine methyltransferase; MTR) and methylmaloyl-CoA

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mutase, respectively,1-3 showing the essential role of vitamin B12 for cellular physiological

13

events. As the coenzyme of MTR, vitamin B12 is associated with DNA methylation for gene

14

regulation

15

S-adenosylmethionine (SAM), and the similarity of symptoms between vitamin B12 deficiency

16

and folate deficiency have been known.4

since

which

MTR

are

is

required

involved

as

in

coenzymes

one-carbon

by

methionine

metabolism

that

synthase

produces

17

The study using neuropathy model rats showed the promotion of nerve regeneration by

18

vitamin B12 administration,5 and the study using choline-deficient rats showed that vitamin B12

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administration normalizes the acetylcholine level in the brain, showing the involvement of

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vitamin B12 in myelinogenesis.6,7 Clinically, the administration of vitamin B12 through oral,

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subcutaneous, intramuscular, intravenous, intranasal and eye-drop routes are now used for the

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restoration of neurological function. In the retina, a study with the primary-cultured rat retinal

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neurons revealed the neuroprotective effect of vitamin B12,8 and an in vivo study with vitamin

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B12-difficient monkeys showed a vision disorder and loss of retinal ganglion cells,9 supporting a 3 ACS Paragon Plus Environment


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reduction in the thickness of the peripapillary retinal nerve fiber layer in patients with vitamin B12

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deficiency.10 The intramuscular administration of vitamin B12 has been reported to suppress the

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retinal exudate production in patients with diabetic retinopathy,11 and the intramuscular

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administration of hydroxocobalamin has been reported to contribute to the recovery of vision in

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tobacco amblyopia.12 Recent study suggested that vitamin B12 functions as a superoxide

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scavenger,13 and the intravitreal injection of cyanocobalamin has been reported to increase the

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survival of retinal ganglion cells by reducing the burst of intracellular superoxide.14

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Cumulative evidence supports the physiological and pathological significance of vitamin

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B12 in the retina, and the supply of vitamin B12 to the retina from the circulating blood has an

10

important role in the maintenance vitamin B12 homeostasis in the retina. In the retina, the neural

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tissue is separated from the circulating blood by two different barrier structures, the inner

12

blood-retinal barrier (BRB) and the outer BRB, and the responsible cells are retinal capillary

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endothelial cells and retinal pigment epithelial (RPE) cells, respectively.15 At these barrier

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structures, tight junctions are formed to restrict paracellular solute transport, and the supply of

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nutrients to the retina from the circulating blood takes place through transporter-mediated

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transport processes,16,17 such as the transport of dehydroascorbic acid, methyltetrahydrofolate,

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biotin and riboflavin mediated by glucose transporter (GLUT1/SLC2A1), reduced folate carrier 1

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(RFT1/SLC19A1), sodium-dependent multivitamin transporter (SMVT/SLC5A6) and riboflavin

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transporters (RFVTs/SLC52A), respectively, expressed in retinal capillary endothelial cells and

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RPE cells.18-21 The contribution of receptor-mediated endocytosis is also suggested to the

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nutrient transport into the retina, since transferrin receptor 1 and 2 are expressed at the BRB

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and involved in the maintenance of the iron concentration in the retina.22-24 Furthermore, at the

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outer BRB, receptors for advanced glycation end products (RAGE) are reported to be involved

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in the retina-to-blood transport of amyloid β,25 and fluorescence confocal microscopy revealed 4 ACS Paragon Plus Environment

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the internalization of low-density lipoprotein receptors (LDLR) the involvement of which is

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suggested in the endocytosis of vitamin K.26,27 Scavenger receptor class B, type I

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(SR-BI)-mediated transport has also been reported for the endocytosis of vitamin E in the inner

4

and outer BRB,28 and all this cumulative evidence supports the important contribution of

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receptor-mediated endocytosis to vitamin transport at the BRB.

6

The membrane transport of vitamin B12 has been investigated in various types of cells,

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and the previous studies have suggested the expression of cubilin, megalin (low density

8

lipoprotein-related protein 2; LRP2) and asialoglycoprotein receptor (ASGP-R) in the intestinal

9

epithelial cells.29-34 Intrinsic factor (IF) secreted by the gastric epithelium is involved in the

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intestinal absorption of vitamin B12. The IF-vitamin B12 complex binds to cubilin in the ileal

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brush-border, and the interaction between cubilin and megalin is suggested to be crucial for the

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endocytosis of the IF-vitamin B12 complex.29-32 In addition, ASGP-R expressed in hepatocytes

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or intestinal epithelial cells is suggested to contribute to the uptake of vitamin B12 formed

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complex with haptocorrin (transcobalamin I).32-34 Regarding the tissue distribution of vitamin B12

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from the circulating blood, in vitro transport studies in various human-derived cells, such as skin

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fibroblasts, erythroleukemia (K562 cells), colorectal adenocarcinoma (SW48 cells) and

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embryonic kidney cell line (HEK293 cells), have suggested the involvement of transcobalamin II

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receptor (TCII-R/CD320/TCblR)-mediated endocytosis in the blood-to-tissue transport of

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vitamin B12.35-41 Therefore, vitamin B12 is required to form a complex with TCII protein, and the

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complex associates with TCII-R at the cell surface, followed by internalization mediated by

21

clathrin-dependent endocytosis. Subsequently, the digestion of the complex by cathepsin L

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takes place in the lysosomes,38 and vitamin B12 is freed to be transported from lysosomes to the

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cytosol by the ATP-biding cassette transporter subfamily D member 4 (ABCD4).42,43 The acidic

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lysosomal interior is essential for the stability and activity of cathepsin L, and it has been 5 ACS Paragon Plus Environment


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suggested that the neutralization of lysosomal pH by lysosomotropic agents including

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chloroquine inhibits intracellular vitamin B12 transport.44,45 Based on TCII/TCII-R pathway,

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vitamin B12 analogues such as [111Indium]-diethylenetriaminepentaacetate adenosylcobalamin

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(111In-DAC) was synthesized to be an imaging tool for TCII-R with an implication that it can

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delineate tumors in their locations. 46-48

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However, little is known about the details of the mechanisms involved in the supply of

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vitamin B12 into the retina across the BRB, and cyanocobalamin uptake was examined in order

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to investigate vitamin B12 transport at the BRB in the present study. Here, fluorescence-labeled

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cyanocobalamin (Cy5-cyanocobalamin) was synthesized, and was used in uptake analyses

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under in vivo and in vitro conditions, since the benefit of fluorescence-labeling has been

11

proposed for the study of vitamin B12 and membrane transport across the BRB

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microscopy was performed after internal jugular vein injection of Cy5-cyanocobalamin for in

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vivo distribution analysis, and an in vitro uptake analysis of Cy5-cyanocobalamin was examined

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in an in vitro model cell line of the inner BRB (conditionally immortalized rat retinal capillary

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endothelial cells/TR-iBRB2 cells).52

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49-51

Confocal

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2. Methods

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2.1. Reagents and animals

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Chemicals of reagent grade were used in the analyses, including the chemical

5

synthesis of fluorescence-labeled compounds, and were obtained commercially. Anti-human

6

CD320/TCII-R rabbit polyclonal antibodies were obtained from Proteintech (Rosemont, IL).

7

The experimental protocol for the analysis, using male ddY mice (6 weeks old) with a body

8

weight of 28-30 g and male Wistar rats (6 weeks old) with a body weight of 160-180 g, were

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designed in accordance with the institutional guidelines recently produced by the University of

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Toyama Animal Care Committee, observing the ARRIVE (Animal Research: Reporting In Vivo

11

Experiments) guidelines and the Association for Research in Vision and Ophthalmology

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(ARVO) Statement. Mice were obtained from Japan SLC (Hamamatsu, Japan), and were

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allowed free access to water and food and kept in a humidity- and temperature-controlled room.

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2.2. Synthesis of Cy5-cyanocobalamin

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Cyanine 5 (Cy5)-labeled cyanocobalamin (Cy5-cyanocobalmin) was synthesized as

17

shown in Fig. 1A. Cyanocobalamin-ribose-5’-O-(6-aminohexylamine) (Mw. 1497) was

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synthesized as an intermediate in accordance with a procedure reported by McEwan et al.53 In

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brief, 1,1’-carbonyldiimidazole (2.6 mg/100 µL DMSO, 16 µmol/100 µL DMSO) and

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cyanocobalamin (5 mg/600 µL DMSO, 3.7 µmol/600 µL DMSO) were mixed by stirring for 30

21

min at 30°C. After adding diaminohexane (3 mg/100 µL DMSO, 26 µmol/100 µL DMSO), the

22

reaction mixture was stirred for 24 h at room temperature, followed by adding ethyl acetate to

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initiate recrystallization. After rinsing by ultrasonic treatment in acetone, the resultant

24

precipitates

were

dissolved

in

water,

followed

by

7 ACS Paragon Plus Environment

isolation

and

purification

of


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cyanocobalamin-ribose-5’-O-(6-aminohexylamine) using high performance liquid spectrometry

2

(HPLC) and electrospray ionization-mass spectrometry (ESI-MS), respectively. HPLC was

3

performed on a reversed-phase column (COSMOSIL 5C18-AR-II column, nacalai tesque, Kyoto,

4

Japan), and cyanocobalamin-ribose-5’-O-(6-aminohexylamine) was eluted with aqueous

5

trifluoroacetic acid (TFA) (0.1%) and CH3CN (including TFA 0.1%) with a linear gradient

6

(10–60%) during 0-50 min at a flow rate of 2.0 mL min-1. UV detection was performed at 350 nm,

7

and peaks of [M+H]+ and [M+2H]2+ were observed at m/z = 1497 and 749, respectively.

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Cy5-cyanocobalamin (Mw. 2121) was synthesized in accordance with a procedure

9

reported by Grissom et al.54 Cyanocobalamin-ribose-5’-O-(6-aminohexylamine) (200 µg/100 µL

10

DMSO, 0.13 µmol/100 µL DMSO) was mixed with Cy5 succinimidyl ester (100 µg, 0.13 µmol)

11

and N,N-diisoproprylethylamine (1.5 µg, 8.6 µmol) and stirred for 24 h at room temperature,

12

followed by adding diethyl ether/dichloromethane (2:1 mix) to initiate recrystallization. After

13

rinsing in diethyl ether/dichloromethane (2:1 mix), the resultant precipitates were dissolved in

14

water, followed by isolation and purification of Cy5-cyanocobalamin by HPLC and ESI-MS,

15

respectively. HPLC was performed on a revered-phase column (COSMOSIL 5C18-AR-II

16

column), and Cy5-cyanocobalamin was eluted with 100 mM triethylammonium acetate buffer

17

(pH 7.0) and CH3CN with a linear gradient (20-70%) during 0-50 min at a flow rate of 2.0 mL

18

min-1. UV detection was performed at 600 nm, and peaks of [M+2H]2+ and [M+3H]3+ were

19

observed at m/z = 1061 and 708, respectively.

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2.3. Confocal microscopy of Cy5-cyanocobalamin in the retina

22

The distribution analysis of Cy5-cyanocobalamin to the retina was conducted by

23

internal jugular vein injection of Cy5-cyanocobalamin. In ddY mice anesthetized with

24

pentobarbital (50 mg/kg), saline containing Cy5-cyanocobalamin (1.5 mg/300 µL) was injected 8 ACS Paragon Plus Environment

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into the internal jugular vein. The mice were decapitated 120 min after injection, and their

2

eyeballs were immediately collected, soaked in phosphate-buffered saline (PBS) containing 4%

3

paraformaldehyde for 3 h, then soaked in PBS containing 30% sucrose at 4°C. The eyes were

4

fixed in the optimal cutting temperature compound (O.C.T. compound, Sakura Tissue-Tek,

5

Tokyo, Japan), and tissue slices (15 µm) were prepared using a cryostat (CM1900, Leica,

6

Wetzlar,

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4',6-diamidino-2-phenylindole

8

Laboratories, Burlingame, CA), to be examined with a confocal microscope (LSM780, Carl

9

Zeiss, Oberkochen, Germany). Excitation wavelengths of 405 nm and 633 nm were used for

10

Germany).

Tissue

slices (DAPI)

mounted and

on

glass

VECTASHIELD

slides

were

mounting

treated

medium

with

(Vector

DAPI and Cy5-cyanocobalamin, respectively.

11 12

2.4. Expression and purification of TCII and GFP-TCII proteins

13

A reverse transcription-polymerase chain reaction (RT-PCR) was carried out to amplify

14

cDNAs of TCII and GFP using specific primers (Supplemental Table 1). TCII cDNA did not

15

include the signal sequence (MELLKALLLLSGVLGALA) located at the N-terminal of the TCII

16

since the sequence was posttranslationally removed.55 KOD FX NEO (Toyobo, Osaka, Japan)

17

was used in cDNA amplification, and pAcGFP-C1 (Takara, Shiga, Japan) and rat kidney total

18

RNA were adopted as templates for cloning GFP and TCII cDNAs, respectively. PCR was

19

carried out with a thermal cycler, GeneAmp PCR system 9700 (Thermo Fisher Scientific,

20

Waltham, MA), for TCII through 35 cycles at 98°C for 30 sec and 68°C for 120 sec, and for GFP

21

through 35 cycles at 98°C for 10 sec, 60°C for 30 sec, and 68°C for 90 sec. After allowing the

22

PCR products to acquire overhanging dA at the 3’-ends using a 10×A attachment mix (Toyobo),

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PCR products were subcloned into pGEM-T easy vector (Promega, Fitchburg, WI), and the

24

PCR products were confirmed by sequence analysis in a DNA sequencer (ABI PRISM 3130; 9 ACS Paragon Plus Environment


Page 10 of 43

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Applied Biosystems). Subsequently, GFP cDNA was cloned into a glutathione S-transferase

2

(GFP)-fusion protein expression vector, pGEX-6P-1 vector (GE Healthcare, Chicago, IL), by

3

means of restriction enzymes, BamH I and EcoR I, to construct pGEX-6P-1/GFP. TCII cDNA

4

was also cloned into pGEX-6P-1 and pGEX-6P-1/GFP by means of restriction enzymes, Sma I

5

and Not I, to construct GST-fusion protein expression vectors, pGEX-6P-1/TCII and

6

pGEX-6P-1/GFP-TCII, respectively.

7

Escherichia coli SHuffle competent cells (New England Biolabs, Ipswich, MA) were

8

transformed by the constructed expression vector, and were cultured in Luria-Bertani (LB) broth

9

(3 L) at 37°C for 8 h, followed by centrifugation for 10 min at 6,900 rpm and 20°C using a

10

J-LITE® JLA-10.500 rotor (Beckman Coulter, Brea, CA). The collected cells were suspended in

11

lysis buffer (10 mM ethylenediaminetetraacetic acid (EDTA), 10 mM ethylene glycol tetraacetic

12

acid (EGTA), 1 mM (phenylmethylsulfonyl fluoride) PMSF, 1%(w/v) Triton-X, and 0.07% (v/v)

13

2-mercaptoethanol), and were disrupted at 1,000 psi by means of FRENCH Pressure Cells

14

(Thermo Fisher Scientific) and this was repeated 5 times. The homogenate was centrifuged for

15

30 min at 16,500 rpm and 4°C using a JA-17 rotor (Beckman Coulter) to collect the supernatant

16

containing GST-fusion protein, GST-GFP-TCII and GST-TCII, in a clean tube, and this was

17

repeated twice. The resultant supernatant was incubated with glutathione sepharose 4B with

18

rotation for 12 h at 4°C, followed by transferring glutathione sepharose 4B to an

19

Econo-Column® chromatography column (Bio-Rad Laboratories, Hercules, CA). In the column,

20

GST-fusion protein was eluted by adding glutathione solution (100 µM glutathione reduced form,

21

50 mM Tris-HCl, pH 9.5), and the eluted solution was dialyzed in Ringer-HEPES buffer (141

22

mM NaCl, 4 mM KCl, 2.8 mM CaCl2, 10 mM HEPES, pH 7.4) for 6 h. GST-fusion proteins were

23

gently rotated to react with PreScission Protease (GE Healthcare) for 4 h at 4°C, followed by

24

incubation with glutathione sepharose 4B for 12 h at 4°C. The protein solution was collected by 10 ACS Paragon Plus Environment

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transferring the solution including glutathione sepharose 4B to an Econo-Column®

2

chromatography column (Bio-Rad Laboratories), and the amount of purified recombinant

3

protein, GFP-TCII or TCII, was determined using a DC protein assay kit (Bio-Rad, Hercules,

4

CA) and a Model 680 Microplate Reader (BIO-RAD). Protein expression and purification were

5

confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after

6

incubating protein samples in sample buffer (125 mM Tris-HCl, 4%SDS, 10% sucrose, 0.6%

7

bromophenol blue, 5% 2-mercaptoethanol, pH 6.8) for 5 min at 98°C.

8 9

2.5. Uptake analysis of Cy5-cyanocobalamin

10

Uptake of Cy5-cyanocobalamin was investigated by reference to previous

11

reports.20,45,56-59 Extracellular fluid (ECF) buffer (112 mM NaCl, 25 mM NaHCO3, 3 mM KCl, 1.2

12

mM MgSO47H2O, 0.4 mM K2HPO4, 10 mM HEPES, 10 mM D-glucose, 1.4 mM CaCl2) was

13

used as an assay buffer, since ECF buffer was designed by referring extracellular fluid and has

14

been used in the study with the in vitro model cell lines of the blood-tissue barriers.52,60-63

15

Cy5-cyanocobalamin was incubated with TCII protein or GFP-TCII protein in ECF buffer for 60

16

min to form a Cy5-cyanocobalamin-TCII protein complex or a Cy5-cyanocobakamin-GFP-TCII

17

protein complex. TR-iBRB2 cells (1 × 105 cell/well), an immortalized rat retinal capillary

18

endothelial cell line, were cultured in 24-well plates coated with collagen I (BD Bioscience,

19

Franklin Lakes, NJ), and an uptake assay was started by adding 200 µL ECF containing

20

Cy5-cyanocobalamin at 37°C. The assay was terminated by adding ice-cold ECF-buffer, and

21

cells were treated with 600 µL 2% Triton X solution (20 mM Tris, 137 mM NaCl, 2% Triton

22

X-100) overnight at room temperature. The fluorescence intensity of Cy5-cyanocobalamin in

23

the cell lysate was determined using SpectraMax i3 (Molecular Device, CA, USA) with an λex of

24

642 nm and an λem of 667 nm, and the amount of cellular protein was determined using a DC 11 ACS Paragon Plus Environment


Page 12 of 43

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protein assay kit (Bio-Rad). The uptake of Cy5-cyanocobalamin by TR-iBRB2 cells was

2

evaluated as the cell-to-medium (C/M) ratio that was calculated using Eq. 1.

3 4 5

C/M ratio = (Cy5-cyanocobalamin in the cells (mol/mg protein) / (Cy5-cyanocobalamin in the medium (mol/µL))

(Eq. 1)

6 7

To visually examine the uptake of Cy5-cyanocobalamin at the inner BRB, TR-iBRB2

8

cells were seeded at 2×104 cells/well on 8-well culture slides coated with collagen I, and an

9

uptake assay was carried out by adding 200 µL ECF-buffer containing Cy5-cyanocobalamin for

10

120 min at 37°C. The assay was terminated by washing cells with ice-cold ECF buffer three

11

times. After fixation of the cells with 4% paraformaldehyde, slides were treated with DAPI and

12

VECTASHIELD mounting medium (Vector Laboratories), to be examined with a confocal

13

microscope (LSM780, Carl Zeiss). Excitation wavelengths of 405 nm, 488 nm and 633 nm were

14

used for DAPI, GFP-TCII protein and Cy5-cyanocobalamin, respectively.

15 16

2.6. Expression analysis of TCII-R

17

The mRNA expression of TCII-R was investigated by RT-PCR as described in previous

18

reports,20 and RT-PCR was carried out using ExTaq DNA polymerase (Takara) and specific

19

primers for the TCII-R (Supplemental Table 2), through 30 cycles at 94°C for 30 sec, 60°C for

20

60 sec, and 72°C for 60 sec. The expression of TCII-R protein in TR-iBRB2 cells and

21

conditionally immortalized rat RPE cells (RPE-J cells) was investigated by Western blot

22

analysis of the crude membrane fraction with a Mini-PROTEAN3 kit (Bio-Rad), Hybound-P (GE

23

Healthcare), a Mini Trans-Blot Electrophoretic Transfer Cell kit (Bio-Rad), an ECL-plus Western

24

Blotting Detection System (GE Healthcare) and a luminescent image analyzer LAS-4000 mini 12 ACS Paragon Plus Environment

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(Fujifilm, Tokyo, Japan), as described previously.63,64 Anti-human CD320/TCII-R rabbit

2

polyclonal antibodies (Proteintech) in blocking buffer (25 mM Tris-HCl, 125 mM NaCl, 1%

3

skimmed milk) were used as the primary antibody, and horseradish peroxidase-conjugated

4

anti-rabbit IgG antibodies were used as the secondary antibody for chemiluminescence protein

5

detection. Cells were cultured on 100 mm dishes, and collected in a clean tube, followed by

6

centrifugation for 5 min at 200×g and 4°C. The resultant pellet was suspended in suspension

7

buffer (1 mM EDTA, 1 mM EGTA, 10 mM HEPES, 320 mM sucrose, pH 7.4) containing

8

protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) and subjected to nitrogen cavitation

9

(850 psi, 4°C, 20 min) by means of a Parr cell disruption bomb (Parr instrument company,

10

Moline, IL), followed by centrifugation for 20 min at 10,000×g and 4°C. The resultant

11

supernatant was centrifuged for 60 min at 100,000×g and 4°C, and a crude membrane fraction

12

was obtained as a pellet, and suspended in suspension buffer containing protease inhibitor

13

cocktail and stored at -80°C.

14

The immunohistochemistry with confocal microscope LSM780 was carried out as

15

reported in our previous reports.63 The frozen sections (thickness 18 µm) was prepared from

16

Wistar rats, and anti-human CD320/TCII-R rabbit polyclonal antibodies (Proteintech) and

17

guinea pig polyclonal anti-GLUT1 antibodies were used as the primary antibodies.65 For

18

confocal microscopy, Alexa Fluor 488-conjugated anti-rabbit IgG antibodies and Alexa Fluor

19

568-conjugated anti-guinea pig IgG antibodies (Thermo Fisher Scientific) were used as the

20

secondary antibody.

21 22

2.8. Statistical analysis

23

Statistical analyses were carried out using a one way analysis of variance (ANOVA)

24

followed by the Bonferroni correction method, respectively. Unless otherwise indicated, all data 13 ACS Paragon Plus Environment


1

represent means ± S.E.M.

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1


3. Results

2 3

3.1. Synthesis of Cy5-cyanocobalamin

4

Cyanocobalamin-ribose-5’-O-(6-aminohexylamine) (Mw. 1497), an intermediate in

5

Cy5-cyanocobalamin synthesis, was synthesized to be purified by means of HPLC, and

6

ESI-MS

7

cyanocobalamin-ribose-5’-O-(6-aminohexylamine) since peaks for [M+H]+ (m/z 1497) and

8

[M+2H]2+ (m/z 749) were detected (data not shown). Similarly, the synthesis of

9

Cy5-cyanocobalamin (Mw. 2121) was also purified by means of HPLC with showing a single

10

peak, and confirmed by the peaks for [M+2H]2+ (m/z 1061) and [M+3H]3+ (m/z 708) detected in

11

the ESI-MS (Fig. 1B and 1C).

analysis

confirmed

the

single

production

of

12 13

3.2. In vivo distribution analysis of Cy5-cyanocobalamin in the retina

14

After the internal jugular vein injection of Cy5-cyanocobalamin, the confocal microscopy

15

detected the fluorescent signals of Cy5-cyanocobalamin (red) in the inner plexiform layer (IPL),

16

the outer plexiform layer (OPL) and the retinal pigment epithelium (RPE) (Fig. 2B-D), while no

17

significant signal was detected in the case of the internal jugular vein injection of saline (Fig. 2A).

18

The signal distribution pattern observed in IPL and OPL suggested the distribution of

19

Cy5-cyanocobalamin in the retinal capillary endothelial cells (Fig. 2B and 2C), since the pattern

20

was similar to the distribution of Griffonia (Bandeiraea) simplicifolia I-Isolectin B4 (GSL

21

I-Isolectin B4), a marker of endothelial cells, that was previously reported.66

22 23 24

3.3. In vitro uptake of Cy5-cyanocobalamin by TR-iBRB2 cells To investigate the blood-to-cell transport of cyanocobalamin at the inner BRB, uptake 15 ACS Paragon Plus Environment


Page 16 of 43

1

analyses were performed in TR-iBRB2 cells which are an in vitro model cell line of the inner

2

BRB. The preincubation of Cy5-cyanocobalamin with TCII protein provided a time-dependent

3

increase of Cy5-cyanocobalamin uptake by TR-iBRB2 cells for at least 60 min, while TR-iBRB2

4

cells showed only a residual uptake of Cy5-cyanocobalamin without preincubation with TCII

5

protein (Fig. 3A). In addition, the prominent uptake of Cy5-cyanocobalamin was observed for

6

150 min, and this supported that Cy5-cyanocobalamin existed to be stable in assay system

7

within 150 min at least. Furthermore, a similar result was also obtained for GFP-fused TCII

8

protein (GFP-TCII protein) (Fig. 3B), and it is suggested that preincubation with TCII protein is

9

essential for Cy5-cyanocobalamin uptake by TR-iBRB2 cells. Assay with GFP-TCII protein

10

exhibited a slightly lower Cy5-cyanocobalamin uptake than that in assay with TCII protein, and

11

this might be caused by fusion with GFP. The inhibitory effect of several compounds on

12

Cy5-cyanocobalamin uptake was investigated by preincubation with TCII protein. The uptake of

13

Cy5-cyanocobalamin by TR-iBRB2 cells was significantly reduced by 44%, 39% and 18% in the

14

presence of unlabeled cyanocobalamin (20 µM), chlorpromazine (100 µM) and chloroquine

15

(100 µM), respectively,45,56,57 while the uptake was unchanged in the presence of L-carnitine

16

(100 µM). The uptake of Cy5-cyanocobalamin was also reduced by 71% in the absence of

17

calcium ion (Ca2+) (Fig. 3C).58,59

18 19

3.4. Intracellular distribution of Cy5-cyanocobalamin in TR-iBRB2 cells

20

GFP-TCII protein was prepared to investigate the intracellular localization of TCII

21

protein, and preincubation with GFP-TCII protein produced a time-dependent increase of

22

Cy5-cyanocobalamin uptake by TR-iBRB2 cells for at least 120 min (Fig. 3B), showing that

23

GFP-fused TCII protein retains the ability to form a complex with Cy5-cyanocobalamin. After

24

incubation of Cy5-cyanocobalamin with GFP-fused protein, the uptake of Cy5-cyanocobalamin 16 ACS Paragon Plus Environment

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1

was investigated by confocal microscopy, and magnified view showed the fluorescence signals

2

of Cy5-cyanocobalamin (red) and GFP-TCII protein (green), exhibiting that they were merged

3

with each other (Fig. 4A, arrows). When a condition with GFP-TCII protein was compared with a

4

condition without GFP-TCII protein in confocal microscopy, the fluorescence signal of

5

Cy5-cyanocobalamin was observed in the presence of GFP-TCII protein (Fig. 4B), while no

6

signal was observed in the absence of GFP-TCII protein (Fig. 4C).

7 8

3.5. Expression analysis of TCII-R

9

Analysis with RT-PCR detected the transcript of TCII-R in TR-iBRB2 cells and RPE-J

10

cells (Fig. 5A). In addition, Western blot analysis with anti-TCII-R antibodies detected TCII-R

11

protein in TR-iBRB2 cells and RPE-J cells (Fig. 5B). In the immunohistochemistry of TCII-R

12

protein in the retina, GLUT1 was used as a marker protein because of its localization at the

13

luminal and abluminal membranes of retinal capillary endothelial cells and at both the apical

14

and basal membranes of RPE cells.63,65 The fluorescence signal of TCII-R (green) was

15

detected in the retinal capillary endothelial cells and RPE cells (Fig. 6A). In the retinal capillary

16

endothelial cells, the signal of TCII-R protein was observed at the luminal and abluminal sides,

17

and was colocalized with GLUT1 (Fig. 6B). In the RPE cells, the signal of TCII-R protein was

18

observed at the apical and basal sides, and was similarly colocalized with GLUT1 (Fig. 6C).

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1

Page 18 of 43

4. Discussion

2 3

Vitamin B12 is involved in various cellular physiological events, such as one-carbon

4

metabolism including folate and methionine metabolism pathways that is associated with DNA

5

methylation.1,2,4 While the intestinal absorption of vitamin B12 has been suggested to involve

6

several molecules, such as IF, haptocorrin, cubilin, megalin and ASGP-R,29-34 the tissue

7

distribution of vitamin B12 involves TCII and TCII-R38-41. In the neural retina, vitamin B12 have a

8

role that is associate to retinal pathological symptoms, and its property as a superoxide

9

scavenger has been also reported.8-14 However, little is known about the details of the

10

mechanisms for supplying vitamin B12 to the retina across the BRB. In the present study, the

11

importance of TCII and TCII-R in the retinal distribution of vitamin B12 was hypothesized, and

12

the in vivo and in vitro visual assessment of cyanocobalamin transport at the BRB was

13

performed, by means of Cy5-cyanocobalamin synthesized successfully (Fig. 1).

14

In the confocal microscopy after injecting Cy5-cyanocobalamin into the internal jugular

15

vein, a possible contribution of the inner and outer BRB was found for cyanocobalamin supply

16

to the retina, since fluorescent signals of Cy5-cyanocobalamin were detected in retinal capillary

17

endothelial cells and RPE cells (Fig. 2). Therefore, the detailed mechanism of cyanocobalamin

18

distribution to the retina at the BRB was investigated in TR-iBRB2 cells, an in vitro model cell

19

line of the inner BRB, since the fluorescence signal of Cy5-cyanocobalamin detected in OPL

20

was stronger than that in RPE (Fig. 2). As the results, the involvement of TCII-R-mediated

21

endocytosis was suggested in cyanocobalamin uptake by the retinal capillary endothelial cells

22

which form the inner BRB, since TR-iBRB2 cells exhibited a time-dependent increase of

23

Cy5-cyanocobalamin uptake after preincubation of Cy5-cyanocobalamin with TCII protein or

24

GFP-TCII protein while the residual uptake of Cy5-cyanocobalamin was observed without 18 ACS Paragon Plus Environment

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1

preincubation with TCII protein (Fig. 3A and 3B), showing that the complex formation of

2

cyanocobalamin and TCII protein is essential for the blood-to-cell transport of cyanocobalamin

3

transport at the BRB. The

4

in

vitro

inhibition

analysis

also

supported

the

involvement

of

TCII

5

receptor-mediated endocytosis in the blood-to-cell transport of cyanocobalamin at the inner

6

BRB, since Cy5-cyanocobalamin uptake by TR-iBRB2 cells was significantly reduced by

7

inhibiting the complex formation of Cy5-cyanocobalamin with TCII protein using unlabeled

8

cyanocobalamin, inhibiting the association of the complex of cyanocobalamin and TCII protein

9

with TCII-R using depletion of Ca2+ ions,58,59 inhibiting clathrin-dependent endocytosis using

10

chlorpromazine,56,57 and inhibiting cathepsin L using chloroquine (Fig. 3C).45 The in vitro

11

inhibition study supported that TCII-R-mediated endocytosis of cyanocobalamin involves two

12

key processes, the association of TCII protein and cyanocobalamin and the association of

13

TCII-R and the cyanocobalamin-TCII protein complex, at least. The strongest inhibition was

14

shown by the depletion of Ca2+ ions, and this implies that the association of TCII-R and the

15

cyanocobalamin-TCII

16

TCII-R-mediated endocytosis of cyanocobalamin. In addition, the present results imply the

17

helpfulness of inhibition approach using anti-TCII and anti-TCII-R antibodies in the further

18

confirmation of TCII/TCII-R pathway, and the utility of Cy5-cyanocobalamin in kinetic study of

19

cyanocobalamin uptake in various types of cells.

protein

complex

may

be

a

relatively

significant

process

in

20

The confocal microscopy of Cy5-cyanocobalamin in TR-iBRB2 cells supported the

21

necessity of TCII protein in cyanocobalamin uptake by the retinal capillary endothelial cells,

22

since

23

Cy5-cyanocobalamin by TR-iBRB2 cells (Fig. 4B and 4C). In addition, the confocal microscopy

24

also suggested that cyanocobalamin exists as a complex with TCII protein in retinal capillary

the

absence

of

GFP-TCII

protein

dramatically


decreased

the

uptake

of


Page 20 of 43

1

endothelial cells, since the intracellular colocalization of Cy5-cyanocobalamin and GFP-TCII

2

protein was observed in TR-iBRB2 cells (Fig. 4A and 4B). Therefore, it is suggested that the

3

complex of cyanocobalamin and TCII protein undergoes vesicular transport in retinal capillary

4

endothelial cells (Fig. 4A and 4B), supporting the involvement of TCII-R-mediated endocytosis

5

in the transport of cyanocobalamin at the inner BRB.67,68

6

The importance of TCII-R-mediated endocytosis at the inner BRB was also supported

7

by the results obtained in the expression analysis, since the expressions of TCII-R were

8

detected in RT-PCR, Western blot and immunohistochemistry (Fig. 5 and 6). The in vivo

9

analysis also suggested cyanocobalamin uptake by RPE cells that is responsible for the outer

10

BRB, and the expression analyses supported that cyanocobalamin transport possibly involves

11

TCII-R-mediated endocytosis at the outer BRB, since the expression of TCII-R was detected in

12

RPE-J cells which are an in vitro model cell line of the outer BRB (Fig. 5). Previously, the

13

immunohistochemical quantification approach in xenograft tumor tissues showed the

14

colocalization of TCII and TCII-R with a greater staining intensity for TCII-R than that for TCII.69

15

In the present study, Figure 6 confirmed the physiological expression of TCII-R, and its

16

colocalization of GLUT1 suggested the expression of TCII-R at the luminal and abluminal

17

membranes of the inner BRB, and at apical and basal membranes of the outer BRB (Fig. 6),

18

supporting the importance of TCII and TCII-R in cyanocobalamin transport across the BRB.

19

TCII-R has a high affinity and specificity for cyanocobalamin-TCII protein complex,70-72

20

and the present in vivo and in vitro analyses supported that the blood-to-cell transport of

21

cyanocobalamin is carried out by a specific transport system, TCII-R-mediated endocytosis, at

22

the blood-side of the BRB such as the luminal membrane of the retinal capillary endothelial cells

23

and the basal membrane of the RPE cells. It is reported that the digestion of the complex by

24

cathepsin L takes place in the lysosomes, and the involvement of ABCD4 has been reported in 20 ACS Paragon Plus Environment

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1

cyanocobalamin transport from lysosomes to the cytosol38,42,43. Regarding the cell-to-retina

2

transport of cyanocobalamin at the retina-side of the BRB such as the abluminal membrane of

3

the retinal capillary endothelial cells and the apical membrane of the RPE cells, an in vitro study

4

previously reported the involvement of transcytosis mediated by TCII-R in the transcellular

5

transport of cyanocobalamin,30,73 and a study with knockout mice reported the cyanocobalamin

6

transport function of ATP-binding cassette subfamily C member 1 (ABCC1, multidrug

7

resistance-associated protein 1; MRP1),74,75 showing the possible contributions of transcytosis

8

and ABCC1 at the retina-side of the BRB.

9

The present study with fluorescence-labeled cyanocobalamin suggested that the

10

blood-to-retina transport of cyanocobalamin across the BRB involves TCII-R-mediated

11

endocytosis, and an understanding of the details of nutrient supply to the retina is expected to

12

contribute to the safe and efficient treatment of the severe retinal diseases. In particular, the

13

high affinity of cyanocobalamin-TCII protein complex for TCII-R has suggested the possible use

14

of this transport system for new drug delivery systems with fewer side effects,70,71,76 and studies

15

with

16

B12-doxorubicin, have been reported.49 The usefulness of receptor-mediated endocytosis has

17

also been proposed for high molecular weight drugs, and vitamin B12 has been reported to be

18

conjugated on the surface of nanoparticles carrying an insulin analogue (Mw. 5,807) or a

19

hepatitis B virus antigen (Mw. 25,420).77,78 Furthermore, the conjugation of drugs with vitamin

20

B12 is expected to improve the treatment of retinal diseases. In particular, in the treatment of

21

age-related macular degeneration (AMD), the intravitreal injection of drugs, such as ranibiumab

22

(Mw. 48,350) and pegaptanib (Mw. ~50,000), is currently performed, and inhibitors of

23

chemokine Rho kinase, such as NR58-3.14.3 (Mw. 1,359) and Rho kinase AMA0428 (Mw. 247)

24

have been investigated for follow-on development.79-81 Therefore, the conjugation of a drug with

vitamin B12-conjugated drugs, such as vitamin B12-methotrexate and vitamin



Page 22 of 43

1

vitamin B12 can possibly help in the use of TCII-R-mediated endocytosis at the BRB for safe and

2

efficient drug delivery into the retina, allowing the i.v. or p.o. drug administration.

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1


Author contributions

2

Yu.K., Yo.K. H.A., M.I. and K.H.: conception and design; Yu.K., R.J., K.N., H.A. and

3

S.A.: collection and assembly of data; Yu.K., R. J., K.N., H.A., Yo.K. and S.A.: data analysis and

4

interpretation; Yu.K., Yo.K. and K.H.: writing manuscript. All authors reviewed the manuscript.

5 6

Acknowledgement

7

This research report was financially supported in part by KAKENHI (grant #

8

JP17K08409 and JP16H05110) from the Japan Society for the Promotion of Science (JSPS),

9

and Research Grants from the Takeda Science Foundation. This collaborative work was also

10

supported by JSPS Core-to-Core Program (B. Asia-Africa Science Platforms).

11 12 13

Additional information The authors declare that they have no conflict of interest.

14 15

Supporting Information

16

Supplemental Table 1. Primers designed for cDNA cloning of TCII and AcGFP

17

Supplemental Table 2. Primers designed for mRNA expression analysis of TCII receptor

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Page 24 of 43

1 2 3

References

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to cyano- and hydroxyl-cobalamin in prevention or treatment of cobalamin deficiency. Mol.

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2. Gille,D.; Schmid, A. Vitamin B12 in meat and dairy products. Nutr. Rev. 2015, 73, 106-15.

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29. Yammani, R.R.; Seetharam, S.; Seetharam, B. Cubilin and megalin expression and their

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interaction in the rat intestine: effect of thyroidectomy. Am. J. Physiol. Endocrinol. Metab.

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2001, 281(5),E900-7.

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30. Bose, S.; Kalra, S.; Yammani, R.R.; Ahuja, R.; Seetharam, B. Plasma membrane delivery,

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1 2 3

Figure legends

4 5

Figure1. Synthesis of Cy5-cyanocobalamin.

6

(A) Synthetic scheme for Cy5-cyanocobalamin conjugate. The primary hydroxyl of the ribose

7

moiety on cyanocobalamin was activated using 1,1’-carbonyldiimidazole (CDI), and

8

1,6-diaminohexane was subsequently attached. Cy5 N-hydroxysuccinimidyl (NHS) ester was

9

conjugated to cyanocobalamin through the 6 carbon linker to form Cy5-cyanocobalamin.

10

Cy5-cyanocobalamin was detected with HPLC (B) and ESI-MS spectrum (C). UV detection was

11

performed at 600 nm, and peaks of [M+2H]2+ and [M+3H]3+ were observed at m/z = 1061 and

12

708. DIEA, N,N-diisopropylethylamine; DMSO, dimethyl sulfoxide.

13 14

Figure 2. In vivo distribution of Cy5-cyanocobalamin in the mouse retina.

15

Confocal images were obtained in GCL-to-CH (A, B), IPL-to-ONL (C) and ONL-to-CH

16

(D) regions. Confocal microscopy of Cy5-cyanocobalamin (1.5 mg/300 µL saline, red) was

17

carried out after injection into the internal jugular vein (B-D). Confocal microscopy was also

18

carried out for the injection of saline (300 µL) without Cy5-cyanocobalamin into the internal

19

jugular vein (A). Left and right images were obtained using different configurations for the

20

signals of Cy5-cyanocobalamin (red) and DAPI (blue) and for the signal of Cy5-cyanocobalamin

21

(red). After injection, mice were decapitated and the eyes were immediately isolated. Nuclei

22

were stained with DAPI (blue). GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner

23

nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OLM, outer limiting

24

membrane; POS, photoreceptor outer segment; RPE, retinal pigment epithelium; CH, choroid. 34 ACS Paragon Plus Environment

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1 2 3

Figure 3. Effect of TCII protein on Cy5-cyanocobalamin uptake by TR-iBRB2 cells. (A)

Time-course

of

Cy5-cyanocobalamin

uptake

by

TR-iBRB2

cells.

4

Cy5-cyanocobalamin (1 µM) was incubated with (closed circles) or without (open circles) TCII

5

protein (2 µM) at room temperature. (B) Cy5-cyanocobalamin (1 µM) was incubated with

6

(closed circles) or without (open circles) GFP-TCII protein (2 µM) at room temperature. An

7

uptake assay was subsequently performed at 37°C. Each point represents the mean ± S.E.M (n

8

= 3). (C) Effect of 20 µM unlabeled cyanocobalamin, 100 µM chlorpromazine, 100 µM

9

chloroquine, 100 µM L-carnitine or Ca2+ was examined on the uptake of Cy5-cyanocobalamin

10

by TR-iBRB2 cells. The effects of Ca2+ and unlabeled cyanocobalamin were examined for 60

11

min, and the effects of chlorpromazine, chloroquine and L-carnitine were examined for 120 min.

12

Each point represents the mean ± S.E.M (n = 4-5). **p < 0.01, *p < 0.05, significantly different

13

from control.

14 15

Figure 4. Confocal microscopy of Cy5-cyanocobalamin and GFP-TCII protein taken up by

16

TR-iBRB2 cells.

17

Cy5-cyanocobalamin (1 µM, red) was incubated with (A, B) or without (C) GFP-fused

18

TCII protein (2 µM, green) at room temperature for 60 min, and uptake assay was performed at

19

37°C for 120 min. Magnified (A) and normal (B) images were obtained for the condition of

20

incubation with GFP-TCII protein. Cells were stained with an intercalant of DNA,

21

4’,6-diamidino-2-phenylindole (DAPI). Arrows show the fluorescence signal merging of

22

Cy5-cyanocobalamin and GFP-TCII protein. Scale bar; 20 µm.

23 24

Figure 5. Expression analysis of TCII-R. 35 ACS Paragon Plus Environment


Page 36 of 43

1

(A) RT-PCR analysis was performed in the presence (RT+)or absence (RT-) of reverse

2

transcriptase. Total RNA sample was extracted from TR-iBRB2 cells and RPE-J cells, and total

3

RNA from rat liver was used as a positive control. (B) Western blot analysis of TCII-R (55 kDa)

4

was performed by using anti-human TCII-R antibodies as the primary antibody. The crude

5

membrane fractions (15 µM) of TR-iBRB2 cells and RPE-J cells were developed on SDS-PAGE,

6

and the crude membrane of rat kidney was used as a positive control.

7 8

Figure 6. Immunohistochemistry of TCII-R in the rat retina.

9

Confocal images were obtained for INL-to-RPE (A), INL-to-OPL (B) and ONL-to-RPE

10

(C). Immunohistochemistry of TCII-R was carried out using anti-TCII-R antibodies (green) and

11

anti-GLUT1 antibodies (red), and the colocalization of both fluorescence appears in yellow or

12

orange. Nuclei were stained with DAPI. Under high magnification, the partial colocalization of

13

TCII-R and GLUT1 proteins was observed in the retinal capillary endothelial cells (B) and RPE

14

cells (C). INL, inner nuclear layer; ONL, outer nuclear layer; OPL, outer plexiform layer; POS,

15

photoreceptor outer segment; RPE, retinal pigment epithelium; TCII-R, transcobalamin II

16

receptor.

17 18 19


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Fig. 1 120x91mm (600 x 600 DPI)

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Fig. 2 231x416mm (600 x 600 DPI)


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Fig. 3 197x489mm (600 x 600 DPI)



Fig. 4 225x369mm (600 x 600 DPI)


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Fig. 5 113x159mm (600 x 600 DPI)



Fig. 6 165x310mm (600 x 600 DPI)


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Graphic Abst 41x16mm (300 x 300 DPI)


Investigation of Receptor-Mediated Cyanocobalamin (Vitamin B12

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