The oligosaccharide moiety of rhodopsin—its structure and cellular location

Neurochemistry

Vol. Pergamon Press Ltd.

I. pp. 245-253. 1980. Printed in Great Britain.

THE OLIGOSACCHARIDE MOIETY OF RHODOPSIN--ITS CELLULAR LOCATION

STRUCTURE AND

H. Shichi*, A. J. Adams* and A. Kobata** *Laboratory of Vision Research, National Eye Institute, National Institutes of Health, Bethesda, MD 20205, U.S.A. **Department of Biochemistry, Kobe University School of Medicine, Kobe, Japan

ABSTRACT The sugar chains of bovine rhodopsin released from opsin by hydrazinolysis were reduced with NaB(3H)4 and fractionated by paper chromatography. Three oligosac3 charides were obtained. The structure of the major ( H)-oligosaccharide (ca. 60% of total) was elucidated by sequential exoglycosidase digestion, methylation analysis and endo-B-N-acetyl glucosaminidase D digestion. The structure of the sugar moiety of rhodopsin was thus identified as: Man. ~ 6

Bl

+4

B +4 1

~ a n

> GlcNac

+ GlucNae

>Asn

GlcNac . . . . ÷ M a n / Since the terminal GlcNac serves as galactose acceptor, the location of the sugar moiety of rhodopsin in the disc membrane was studied by incorporation of (3H)-galactose

(from UDP-(3H)-galactose)

into the disk membrane. After inversion of disks

by freeze-thawing, rhodopsin in the membrane incorporated one mole of (3H)-ga- 3 lactose per mole purified pigment. Intact disks incorporated lower amounts of ( H ~ galactose. It was therefore concluded that the sugar moiety of rhodopsin is located on the internal surface of disk membrane. The results of lectin binding studies are consistent with the conclusion. Inverted disks, but not intact disks, bind concanavalin A (specific for ~-mannosyl residue) and wheat germ lectin (specific for N-acetylglucosamine). Since intact sealed rod outer segments bind concanavalin A, the sugar moiety of rhodopsin in the plasma membrane is probably exposed on the external surface of the rod.

KEYWORDS Bovine rhodopsin; structure of oligosaccharide moiety; cellular location; galactose incorporation; concanavalin A binding.

245

246

H. Shicki, A. J. Adams and A. Kobata INTRODUCTION

The glycoprotein nature of tile rod intrinsic membrane proteill rhodopsin was first noted about 10 years ago (Heller, 1968; Shiehi, Lewis, Irreverre and Stone, 1969). The carbohydrate composition of the oligosaccharide moiety of rhodopsi~ was studied by several workers (Heller and Laurence, 1970; Plantner and Kean, 1976; Fukuda, Papermaster and Hargrave, 1977). The published results unanimously show the presence of mannose and N-acetylglucosamine as its constituents but differ in tbemolar ratios of these two sugars. Heller and Lawrence (1970) have reported that bovine rhodopsin contains 3 moles each of the two sugars per mole, whi]e Plantner and Kean (1976) have found 7 to 9 moles of mannose and 4.5 to 6 moles of N-acetylglucosamine per mole of pigment. A glycopeptide of 16 amino acid residues containing two asparagine-linked sugar chains was isolated from tryptic digests (,f bovine rhodopsin (Hargrave, 1977). Although this confirmed the presence of two o]igosaccharido moieties per rhodopsin molecule, the chemical stmlcture of the chains remains yet to be elucidated. We have therefore attempted to determine the complete structure of the sugar moieties of rhodopsin. We have then used the structura] information fo~7 studies of the spatial orientation of rhodopsin in rod membranes.

MATERIALS AND METHODS Bovine rhodopsin was purified on a column of calcium phosphate-Ce!ite by themethod described previously (Shichi and co-workers, 1969). The sugar chains were liberated from opsin by hydrazinolysis and reduced with NaB(3H)4 after N-acetylation.

A de-

tailed account of the procedure as well as of paper chromatography, exoglycosidase digestion, methylation analysis and endo-B-N-acetylglucosaminidase D digestion will be found elsewhere (Liang and co-workers, 1979). To prepare inverted disks, sealed outer segments were prepared by centrifugation in a continuouS metrizamide gradient (Adams, Tanaka and Shichi, 1978) and disks were released in 5% Ficol] by the method of Smith, Stubbs and Litman (1978). Disks were then inverted by freeze-thawing and separated from intact (uninverted) disks by affinity chromatography on concanavalin A (Adams and co-workers, 1978). GLycosylation of disks was carried out with bovine 3 milk galactosyltransferase (Sigma) and ( H)-UDP-galactose (Adams, Somers and Shic[ {, 1979). For lectin binding studies, fluorescent isothiocyanate (FITC)-]abeled lectins (Miles Labs.) were used. Disks were incubated with either FITC-concanavalin A, FITC-wheat germ agglutinin or FITC-Ricinus Communis agglutinin 60 for 20 minutes at 20°C and centrifuged at 58,000 xg for 20 minutes. The f]uorescence of the supernatant was determined with an Aminco-Bowman spectrophotofluorometer. Disks were also incubated in a similar manner in the presence of appropriate inhibitor (~-methylmannoside for conoanavalin A, N-acetylglucosamine for wheat germ agglutinin, and galactose for Ricinus lectin).

RESULTS AND DISCUSSION It would be helpful to have a quick look at the proposed structures of rhodopsin sugar moieties (Fig. 2) before evidence for the structures is discussed. The structures possess one N-acetylglueosamine (at the reducing end) which is linked to an asparagine residue and another N-acetylglucosamine (at the non-reducin~ end) which is linked to the mannose cluster. Acid hydrolysis followed by N-acetylation of the reduced oligosaccharides yielded N-acetylglucosaminitol as the only radioactive sugar. Therefore, the reducing termini of the oligosaccharides must be N-acetylglucosamine. Paper chromatography of the NaB(3H)4-reduced oligosaccharides

gave rise to three fractious,

A, !~, and C in

Oligosaccharide Moiety of Rhodopsin

247

a molar ratio of 55:12:33 (Fig. I). Carbohydrate compositions of the three oligosaccharides are shown in Table I. Since the N-acetylglucosamine at the reducing termini of the samples was already reduced with NaBH4, glucosamine values in Table 1 are l mole less than the actual numbers. The corrected numbers are shown in parentheses. i

i

I

I

I

!

I

" 0

10 20 DISTANCE FROM ORIGIN (cm)

30

Fig. i. Paper chromatography of radioactive oligosaccharides liberated from bovine rhodopsin by hydrazinolysis and subsequent reduction with Nab(3H) 4" TABLE l

Monosaccharide Composition of Oli$osaccharides A, B and C

Oligosaccharides

Molar ratio Mannose

A B C

3.15 4.10 4.92

N-acetylglucosamine 2.00 (3.00)* 2.00 (3.00)* 2.00 (3.00)*

*Numbers in parentheses are corrected integers obtained by adding i mole of N-acetylglucosamine located at the reducing termini. After removal of one N-acetylglucosamine at the non-reducing end by digestion with 6-N-acetylhexosaminidase, oligosaccharides A, B, C became susceptible to ~-mannosidase and released2, 3 and 4 mannose residues, respectively. The remaining triitols were identical with ManBl+ 4GIcNAc61+ 4GlcoINac. This was further confirmed by sequential digestion with 6-mannosidase and N-acetylhexosaminidase. These results indicate that the structure of oligosaccharides A, B, and C can be written as GIcNAc~ (Man~)n.ManBl+ 4GIcNAcBI+ 4GIcNAc, where n=2 for A, n=3 for B, and n=4 for C. In order to determine the location of each glycosidic linkage, oligosaccharides A, B, and C were subjected to methylation analysis (Table 2). The data in Table 2 confirmed that oligosaccharides A and B contain one 3,6 disubstituted mannose and C contains two of it. When de-N-acetylglucosaminyl oligosaccharides obtained by 6-N-acetylhexosaminidase treatment of oligosaccharides A, B and C were subjected to methylation analysis, 3, 4, 6-tri-0-methylmannitol acetate

N.C.I. 1/I-4 P

0 0.9

1.0

1.0

1.0

0.9

0

0

0.9

0

1.1

0.8

0

0

0.8

0.9

0.9

1.0

0.8

1.0

0.2

i.i

0.9

0

0.9

1.0

0.8

0

0.2

1.8

(a) Numbers were calculated by making the values of 2,4-di-0-methylmannitol

1,3,5,6-Tetra-O_-methyl(4-mono-0_-acetyl) 3,4,6-Tri-0-methyl(l,5-di-0--acetyl) 3,6-Di-0_-methyl(l,4,5-tri-O_-acetyl)

2-N-Methylacetamido2-deoxyglucitol

2.1

1.0

~GIcNAc

Intact

Intact

-GIcNAc

.Oligosaccharide B

ratio (~)

O_li$osaccharide A

Molar

1 .0

0

0 .8

2.0

0

0

0

2.8

-GIcNAe

as either 1.0 or 2.0

0.9

l.O

0.9

2.0

0

0.9

0

1.9

Intact

O ligosaccharide C

Molar Ratio of Aldito! Acetates Obtained from Hydrolysates of Permethylated Oli$osaccbarides

2,3,4,6-Tetra-O-methyl(l,5-di-0-acetyl) 2,4,6-Tri-0--methyl(l,3,5,-tri-O_-acetyl) 3,4,6-Tri-0-methyl(l,2,5-tri-0_-acetyl) 2,3,4-Tri-0--methyl(l,5,6-tri-0--acetyl) 2,4-Di-O-methyi(l,3,5,6-tetra-0--acetyl)

Mannitol

TABLE 2

o

>

0~

>

>

co

Fig.

2.

Proposed

structures

of o l i g o s a c c h a r i d e s

A,

B

and

C.

Manel~ 6 _Mane Manel J3 I~6Man~I÷4GIcNAcBI+4GIcNAc GIcNAcBI-2Man~173

Oligosaccharide C

Manel~3 Ow 6Manel~ 6 GlcNAcSl+2Mane113 Man~l+4GlcNAcSl+4GlcNAc

Oligosaccharide B

Manel~ 6 oMan~I÷4GIcNAcSI+4GIcNAc GlcNAcSl+2Man~l ~a

Oligosaccharide A

D" O o ~o

O ~h

O

m o

o

O

250

H. Shicki, A.

Adams and A. Kobata

J.

Manal-2Man~l~6 3Mansl~ Manul~2Man~l I ~ManSl~4R ~Glc~Glcl~3Glcl*3Man~l~2Manol~2Man~113 2 Glucose~

Step 1

4 Mannose~-S I

Step 2

Manul~6.~ ~Manal%. ManulJ° ~ManSl~4R Manul-~

UDP-GlcNAc~ UDP ~

Step 3

Manulk 6 3Mansl~. Manul p ~ManSl*4R GIcNAcSI~2Manul ~

B©

1

Man~l~3 ~ 6Man~l~6 GlcNAcSl~2Manalj3ManSl*4R

Step 4

1

Manal~& -ManSl~4R GlcNAcSl÷2Manul ~3'

UDP-GlcNAc-~ UDP ~

]

Step 5

GIcNAcSI~2MansI~6 3ManSl~4R GlcNAcSl*2Mansl F UDP-Gal UDP ~

1 Step 6

GalSl~4GlcNAcSl~2Man~11~3ManSl~4R GalSl*4GlcNAcSl~2Mansl R=GlcNAcSl~4G]cNAc~Asn-peptide Fig. 3. Processing in the biosynthetic pathway of complex type asparagine-linked sugar chains of glycoprotein.

Oligosaccharide Moiety of Rhodopsin

25|

disappeared with the concomitant appearance of i mole of 2,3,4,6-tetra-O-methyimannitol acetate in all three oligosaccharides. Therefore, the non-reducing terminal N-acetylglucosamine residues in all three oligosaccharides should occur as GlcNAc$1+2Man grouping. These results, taken together, lead us to conclude that oligosaccharides A, B, C have chemical structures shown in Fig. 2. A biosynthetic pathway of complex type asparagine-linked sugar chains of glycoprotein has recently been proposed by Kornfeld, Li and Tabas (1978) (Fig. 3). The proposed pathway indicates that the mannose-rich oligosaccharide moiety is de-mannosylated in a stepwise fashion and then glycosylated (i.e., galactosylated, sialylated, and fucosylated). If the sugar moiety of rhodopsin is processed by the proposed pathway, oligosaccharide C would be de-mannosylated one at a time in two steps to oligosaccharide A. Since oligosaccharide A is the major fraction produced by hydrazinolysis of opsin, the structure is considered to be the final form of rhodopsin sugar moiety. This suggests that the inner segment of bovine rod, where processing of the sugar moiety occurs, lacks glysosyltransferases such as galactosyl transferase and sialyltransferase which are involved in further modification of oligosaccharides. The structure of oligosaccharide A containing mannose~l+3 and mannose~l+6 linkages is consistent with the previous finding (Adams and co-workers, 1978) that the glycoprotein rhodopsin is capable of binding concanavalin A. The N-acetylglucosamine at the non-reducing end of the oligosaccharide moiety of rhodopsin should be able to serve as a galactose acceptor for UDP-galactose: glycoprotein galactosyl transferase (EC 2.4.1.22). We took advantage of the structural feature to study the spatial arrangement of rhodopsin in the disk membrane. After inverted disks were incubated with UDp(3H)-galactose and galactosyltransferase, rhodopsin was extracted and purified on an ECTEOLA-cellulose column. The first peak from the column is non-phosphorylated rhodopsin and the second peak is phosphorylated rhodopsin (Shichi and Somers, 1978).(3H)-Galactose was incorporated into both rhodopsinfractions. The stoichiometry of incorporation was 0.92 galactose per rhodopsin. If rhodopsin has two carbohydrate moieties (Hargrave, 1977), only one of them might be available as galactose acceptor. Intact (uninverted)disks, when similarly treated, incorporated 0.6 galactose per rhodopsin. This low significant level of incorporation may be attributed to vesiculated disks which took up UDP-galactose and transferase during membrane resealing without inversion. Shaper and Stryer (1977) demonstrated incorporation of galactose into the carbohydrate moiety of rhodopsin but did not answer the question on the localization of sugar moiety in the membrane. In our work, galactose-labeled inverted disk vesicles were first separated, rhodopsin extracted, and then the presence of galactose in purified rhodopsin was demonstrated. Leetin binding studies (Table 3), show that inverted disks but not intact disks bind not only concanavalin A but also wheat germ agglutinin which are specific for ~-mannosyl residue and N-acetylglucosamine, respectively (Lis and Sharon, 1973). In contrast, Ricinus lectin that is specific for galactose (Lis and Sharon, 1973) did not bind to disks both intact and inverted. The results are consistent with the lectin binding property of rhodopsin that the chemical structure of the carbohydrate moiety would predict. We therefore conclude that the carbohydrate moiety of the pigment is exposed on the internal (intradiscal) surface of the disk. We have previously shown that intact sealed rod outer segments bind concanavalin A (Adams and co-workers, 1978). If the lectin binding is attributed to the sugar moiety of rhodopsin associated with the plasma membrane (Jan and Revel, 1974; Basinger, Bok and Hall, 1976), the results support the conclusion that the disks are formed by infolding of the rod plasma membrane; the membrane is inverted during the infolding process.

*Fluorescence

B i n d i n g to D i s k s

6.0 6.1 4.0 4.3

Fresh disk - inhibitor Fresh disk + inhibitor Frozen disk - inhibitor Frozen disk + inhibitor

FITCRicinus

is in a r b i t r a r y units.

lectin

4.1 6.5 1.9 4.6

Fresh disk - inhibitor Fresh disk + inhibitor Frozen disk - inhibitor Frozen disk + inhibitor

FITCwheat germ agglutinin

Fluorescence of s u p e r n a t a n t *

5.9 6.6 1.7 5.0

Sample

Lectin

~re~h disk - inhibitor Fresh disk + inhibitor Frozen disk - inhibitor Frozen disk + inhibitor

3

FITCconcanavalin A

Lectin

TABLE

©

>

>

>

~o L~ bo

Oligosaccharide Moiety of Rhodopsin

253

REFERENCES Adams, A. J., R. L. Somers, and H. Shichi (1979). Spatial arrangement of rhodopsin in the disk membrane as studied by enzymatic labeling. Photochem. Photobiol., 29, 687-692. Adams, A. J., M. Tanaka, and H. Shichi (1978). Concanavalin A binding to rod disks. Exp. Eye Res.~ 27, 595-605. Basinger, S., D. Bok, and M. Hall (1976). Rhodopsin in the rod outer segment plasma membrane. J. Cell Biol., 69, 29-42. Fukuda, M., D. S. Papermaster, and P. A. Hargrave (1977). Carbohydrate moiety of bovine rhodopsin. Fed. Proc.~ 36, 895. Hargrave, P. A. (1977). The aminoterminal tryptic peptide of bovine rhodopsin. A glycopeptide containing two sites of oligosaccharide attachment. Biochim.Biophys. Acta~ 492, 83-94. Heller, J., and M. A. Lawrence (1970). Structure of the glycopeptide from bovine visual pigment 500. Biochemistry~ 9, 864-869. Heller, J. (1968). Structure of visual pigments. I. Purification, molecular weight, and composition of bovine visual pigment 500. Biochemistry, 7, 2906-2920. Jan, L. Y., and J. P. Revel (1974). Ultrastructural localization of rhodopsin in the vertebrate retina. J. Cell Biol.~ 62, 257-273. Kornfeld, S., E. Li, and I. Tabas (1978). The synthesis of complex-type oligosaccharides. J. Biol. Chem., 253, 7771-7778. Liang, C. J., K. Yamashita, C. G. Muellenberg, H. Shichi, and A. Kobata (1979). Structure of the carbohydrate moieties of bovine rhodopsin. J. Biol. Chem., 254, 6414-6418. Lis, H., and N.Sharon (1973). The biochemistry of plant lectins (phytohemagglutinins).Ann. Rev. Biochem.~ 42, 541-574. Plantner, J. J., and E. L. Kean (1976). Carbohydrate composition of bovine rhodopsin. J. Biol. Chem., 251, 1548-1552. Shaper, J. H., and L. Stryer (1977). Accessibility of the carbohydrate moiety of membrane-bound rhodopsin to enzymatic and chemical modification. J. Supramol. Struct., 6, 291-299. Shichi, H., M. S. Lewis, F. Irreverre, and A. I. Stone (1969). Biochemistry of visual pigments. I. Purification and properties of bovine rhodopsin. J. Biol. Chem., 244, 5 2 9 - 5 3 6 . Shichi, H., and R. L. Somers (1978). Light-dependent phosphorylation of rhodopsin. Purification and properties of rhodopsin kinase. J. Biol. Chem.~ 253, 7040-7046. Smith, H. G., G. W° Stubbs, and B. J. Litman (1978). The isolation and purification of osmotically intact disks from retinal rod outer segments. Exp. Eye Res., 20, 211-217.