The amino-terminal tryptic peptide of bovine rhodopsin A glycopeptide containing two sites of oligosaccharide attachment

Biochimica et Biophysica Acta, 492 (1977) 83-94

© Elsevier/North-Holland Biomedical Press BBA 37645 T H E A M I N O - T E R M I N A L T R Y P T I C P E P T I D E OF BOVINE R H O D O P S I N A G L Y C O P E P T I D E C O N T A I N I N G TWO SITES OF O L I G O S A C C H A R I D E ATTACHMENT

PAUL A. HARGRAVE School of Medicine and Department of Chemistry and Biochemistry, Southern Illinois University, Carbondale, 111. 62901 (U.S.A.)

(Received November 19th, 1976)

SUMMARY A glycopeptide (T1) composed of 16 amino acids has been isolated from a tryptic digest of bovine rhodopsin. Its sequence is Met-Asn(CHO)-Gly-Thr-Glu-GlyPro-Asn-Phe-Tyr-Val-Pro-Phe-Ser-Asn(CHO)-Lys. Both rhodopsin and peptide T1 are blocked at their amino terminals. When a method specific for isolating aminoterminal tryptic peptides from proteins is applied to rhodopsin, peptide T1 is demonstrated to be the amino-terminal peptide of rhodopsin. Peptide T1 contains two sites at which carbohydrate is attached, whereas rhodopsin was previously thought to contain only a single such site.

INTRODUCTION Rhodopsin is the retinal-containing protein in rod cells of the vertebrate retina which is responsible for dim light and black-and-white vision. It is the principal protein of rod cell outer segment membranes, comprising 85 ~ 5 ~o by weight of the membrane protein [1-4]. Only recently has rhodopsin been prepared in a state of high purity [5-8]. It has not yet been well characterized from any source. Molecular weights ranging from 28 000 [5, 6, 9] to 40 000 [4, 10] have been reported for both bovine and frog rhodopsin. Recent studies fall in the range 35 000-39 000 [1, 11, 12]. Rhodopsin contains a high proportion of hydrophobic amino acids and is a glycoprotein containing both mannose and glucosamine [7, 13]. The composition of a retinal-linked peptide has been reported for bovine rhodopsin [14] as well as the sequence of a nine-amino acid glycopeptide [13]. Both the amino- and carboxylterminal amino acids have been reported to resist characterization by usual end-group procedures [5, 15]. * Letters which designate the mode of chemical or enzymatic generation of the peptide are C -chymotrypsin, C B - cyanogen bromide, P - papain, S = subtilisin, T = trypsin. The number following the letter designates the order of elution of the peptide from a column, or its mobility ranking (if prepared by electrophoresis, the lowest number is assigned to the peptide of greatest mobility).

84 This paper reports the isolation and amino acid sequence of the blocked aminoterminal tryptic peptide of bovine rhodopsin. This peptide has been found to contain the nine-amino acid glycopeptide described by Heller and Lawrence [13] in aminoterminal blocked form, as well as a new second site of carbohydrate attachment. MATERIALS AND METHODS

Materials. Analytical reagent grade chemicals and solvents were used without further treatment unless otherwise specified. Hydroxylamine hydrachloride was recrystallized from methanol; hydrocinnamic acid was recrystallized from ethanol/ ether. Formic acid (Eastman, practical, 99~) was distilled before use. Deionized urea was freshly prepared by passing a 10 M stock solution of urea over a column containing BioRad AG 501-X8 mixed-bed ion-exchange resin. Diisopropylfluorophosphate (Sigma) was prepared as a 50~ solution in anhydrous isopropanol and stored in small aliquots at ....20 °C. Ammonyx LO, a 30 ~ solution of mostly lauryldimethylamine oxide, was the gift of Onyx Chemical Co., Jersey City, N.J. Dodecyltrimethylammonium bromide was prepared as described by Hong and Hubbell [16]. Carboxypeptidase B (Code COBC, 210 units/mg; Worthington) was stored frozen as small aliquots which were treated with diisopropylfluorophc.sphate immediately prior to use. Chromatographically pure /~-trypsin was prepared from twice crystallized bovine trypsin (Worthington)[17]. Chymotrypsin (Worthington, 3x crystallized), further purified by chromatography on Sephadex G-75, was the gift of Dr. Jerry Slightom. Both trypsin and chymotrypsin were prepared immediately prior to use as stock solutions in ice-cold 1 mM HCI. Acid hydrolysis of peptides, proteins, and aliquots from column fractions, was performed in an evacuated glass chamber containing HCI [18]. Amino acid analyses were performed with a Beckman 120C amino acid analyzer employing a dual-column analysis system, or with a Durrum D500 analyzer employing a single column. For semiquantitative amino acid analysis, samples were analyzed by one-dimensional high voltage paper electrophoresis [18]. Ion-exchange chromatography of peptides was performed with BioRad AG50W-X8 resin, 200-400 mesh (BioRad, Richmond, Calif.) washed according to the procedure of Schroeder [19]. Preparation of rhodops&. Rod outer segment membranes were prepared under dim red light from 400 frozen dark-adapted bovine retinas (Geo. Hormel and Co., Austin, Minn.) by the method of Papermaster and Dreyer [12]. The membranes banding at the sucrose 1.11-1.13 g/ml interface were harvested, diluted and pelleted. Unless used immediately, membranes were suspended in buffered sucrose (1.10 g/ml) diluted 1:2 with water, and stored frozen at --20 °C. The rod outer segment membrane pellet was homogenized with a glass-glass homogenizer in Tris buffer (10 mM Tris/acetate, pH 8.0, 5 mM NazEDTA), diluted with the same buffer, and pelleted at 27 000 x g for 30 min at 4 °C. The rod outer segment pellet was dissolved by stirring overnight at 4 °C in 5 ~ Ammonyx LO containing 5 mM Tris/acetate buffer (pH 7.8), 1 mM in NazEDTA and dithioerythritol. The clear supernate, approx. 15 ml obtained after centrifuging at 35 000 × g for 40 min, was submitted to gel filtration in 0.3 ~ Ammonyx LO containing the same buffer as described previously [8]. The 498 nm absorbing rhodopsin

85 peak had an optical ratio 280 nm/498 nm of 1.6 and consisted of a single polypeptide chain when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [8]. After dialysis vs. several changes of deionized water for 48 h, the rhodopsin was made 100 mM in hydroxylamine hydrochloride and 67 mM in phosphate buffer (pH 7.0) and exposed to light until the protein solution was colorless. The hydroxylamine treatment converts the released all-trans-retinal to the oxime, preventing its possible non-specific reaction with amino groups of the protein. The suspension was also made 1 mM in Na2EDTA in order to avoid possible oxidative side reactions of hydroxylamine [20]. The suspension was concentrated by ultrafiltration using an Amicon ultrafiltration cell with a PM-10 membrane. Rhodopsin was also prepared by chromatography on hydroxyapatite in dodecyltrimethylammonium bromide, with minor modifications [16]. We used rod outer segment membranes from 200 retinas, without lyophylization, and employed a 2.5 × 8.0 cm chromatographic column. Sodium phosphate buffer was reduced to 7.5 mM, and dithioerythritol was substituted for dithiothreitol. Reduction and aminoethylation of rod cell membranes, or chromatographically purified rhodopsin, was performed in 8 M urea [21]. Following ethyleneimine treatment, excess reagent was reacted with fl-mercaptoethanol prior to acidification with acetic acid in order to avoid side reactions [22]. After dialysis vs. several changes of dilute acetic acid for 48 h, the protein was lyophilized. The dry residue was extracted with several 20-ml portions of 2:1 (v/v) chloroform/methanol containing a few drops of concentrated NH4OH, to remove retinal-oxime and phospholipids. (Lipid extraction was omitted with dodecyltrimethylammoniumbromide-purified rhodopsin). The white residue was washed on Whatman No. 1 filter paper with absolute ethanol, followed by portions of 95, 75, 50, and 25 % ethanol, and finally deionized water. Amino-terminal analysis, peptide preparation and sequencing. Aminoethylrhodopsin was dansylated by the procedure of Gray [23]. Homogeneity of peptides was determined by electrophoresis at pH 1.7 [18] and at pH 3.5 [24], by quantitative amino acid analysis, and by dansylation [25]. Peptides were sequenced by the dansylEdman procedure [25]. Dansyl amino acids were identified by chromatography on Cheng-Chin polyamide plates (Pierce Chem. Co.) as described by Hartley [26]. The amide status of peptides was determined by electrophoresis at pH 6.5 [24]. Small neutral, acidic and basic peptides (Phe-Ala, Trp-Glu, Arg-Phe) were employed as standards. Peptide mixtures which were to be purified by preparative paper electrophoresis were applied to a 180 cm sheet of Whatman 3 Chroma electrophoresis paper and electrophoresed in 6.7% formic acid for 0.5 h at 1000V followed by 3 h at 5000 V using a Gilson Model DW electrophorator. After drying, peptides were visualized with the fluorescamine spray reagent [27] and eluted from the paper strips with dilute formic acid [24]. Aliquots were hydrolyzed and the remainder of the eluted peptides stored frozen at --20 °C. Preparation of the tryptic glycopeptide. This peptide has been prepared on a large scale several times both from chromatographically purified rhodopsin and from rod cell membranes. The reduced-aminoethylated and delipidated material, containing 1-1.2 g protein by amino acid analysis, was suspended in approx. 50 ml 100 mM NHaHCO3 using a glass-glass homogenizer. This suspension was incubated with 10 mg trypsin and incubated overnight. Digestion was terminated with diiso-

86 propylfluorophosphate and the suspension centrifuged (27 000 × g, 20 min). The pellet was suspended in water by vortex mixer and centrifuged, and the pooled supernates were lyophilized. The lyophilized peptides were suspended in 10-15 ml of 50 mM pyridine acetate buffer (pH 2.4), the pH adjusted to pH 2.2-2.4 with formic acid, and insoluble material removed by centrifugation. The soluble peptides were chromatographed on a column of AG50 resin. In experiments designed solely for preparation of the glycopeptide, the column was eluted at room temperature with just the pH 2.4 buffer. Glycopeptide-containing fractions were pooled and further purified by Biogel P6 chromatography. The glycopeptide was obtained in pure form following chromatography on a 0.9 x 25 cm column of AG50 resin equilibrated in formic acid (pH 2.6). Column effluents of all steps were monitored at 280 nm. Aliquots from alternate tubes were analyzed by acid hydrolysis followed by paper electrophoresis in order to detect the glucosamine-containing peptide. Digestion of the tryptic glycopeptide. Glycopeptide TI, 4 #mol/ml in 100 mM NH4HCO3, was digested with 60/~g/ml chymotrypsin at 37 °C for 2 h. The reaction mixture was quenched with diisopropylfluorophosphate and lyophilized. The digest was dissolved in dilute formic acid (0.2 ~, v/v), adjusted to pH 2.4, and the solution applied to a 0.9 × 15 cm column of AG50 resin equilibrated in the same solvent. Ten 2-ml fractions were collected, then the column was eluted with 4 M NH4OH. The ammonia eluate was lyophilized, and further analyzed by Biogel P6 chromatography in 5 ~ acetic acid. Peptide T1-C1 was incubated at 2.2 #mol/ml with 140/~g/ml papain, prepared essentially according to Brockelhurst et al. [28]. After digestion for 23 h at 37 °C, the digestion mixture was separated by Biogel P6 chromatography in 5 ~ acetic acid. Subtilisin (Carlsberg type, Novo Enzyme Corp.), 250/~g/ml, was used to digest 1.75/zmol peptide T1-C1, 1.75 #mol/ml, in 100 mM NH4HCO3 at 37 °C for 5 h. The reaction was terminated with' diisopropylfluorophosphate. The products were separated by gel filtration (1.0 x 220 cm column of Biogel P6 equilibrated in 100 mM NH4HCO3; fractions were monitored at 225 nm). Cyanogen bromide cleavage followed the procedure of Steers et al. [29]. The peptide, 1.1 mM, in 70 ~ formic acid, was treated at room temperature for 24 h with 80 mM CNBr (Eastman). The reaction mixture was rotary evaporated, dissolved in 5 ~ acetic acid and separated on a column of Biogel P6 equilibrated in the same solvent. Column effluent was monitored by amino acid analysis of acid-hydrolyzed aliquots. Peptide T1-C2 was digested with aminopeptidase M (Sigma) in 100 mM Nethylmorpholine acetate (pH 7.4) 5 mM in MgClz at 37 °C for 18 h (enzyme concentration 500 #g/ml; peptide concentration 3/~mol/ml). The peptide was recovered free from digested amino acids by chromatography on a 1.0 × 220 cm column of Biogel P6 equilibrated in 100 mM NH4HCO3. Peptide was detected by absorbance at 225 rim. Amino-terminal peptide isolation from suecinyl-aminoethyl rhodopsin. Lyophilized aminoethyl-rhodopsin was suspended in freshly deionized 10 M urea. The suspension, 6 mg/ml in protein, was adjusted to pH 8.4 with N-ethylmorpholine and made l0 ~ in acetonitrile prior to addition of succinic anhydride to a final concentration of 100 mM. The pH was kept at 8.0-8.5 with N-ethylmorpholine during the 45 min reaction. The resulting clear solution of succinyl-aminoethyl-rhodopsin was dialyzed vs. several changes of deionized water adjusted to pH 8.3 with N-ethyl-

87 morpholine. Following concentration by ultrafiltration using an Amicon PM-10 membrane, the solution of 38.6 mg of protein (determined by amino acid analysis) was adjusted to pH 8.1 with N-ethylmorpholine and digested with 7 ~ (w/w) fl-trypsin. After 6.5 h, the reaction was stopped by addition of diisopropylfluorophosphate. The digestion mixture was made 1 mM in hydrocinnamic acid (a carboxypeptidase A inhibitor), readjusted to pH 8.1 with N-ethylmorpholine and 300 units of diisopropylfluorophosphate-treated carboxypeptidase B were added. After 1 h of incubation at 37 °C, the reaction mixture was chilled and centrifuged (27 000 x g for 20 min). The supernatant was loaded on a 1.0 x 208 cm precalibrated column of Biogel P2 (50-100 mesh) equilibrated in 100 mM NH4HCO3. The peptide fraction (monitored at 230 and 280 nm) appeared in the 46 ml to 62 ml eluate, well separated from the bulk of small molecules such as hydrocinnamic acid and free arginine (insuring low ionic strength for the following ion-exchange chromatography step). Following lyophilization, the peptides were dissolved in about 4 ml water and titrated to pH 2.6 with formic acid. Following centrifugation, the soluble peptides were loaded on a 0.9 × 25 cm column of AG50 resin equilibrated in formic acid (pH 2.6) which was eluted with the same solvent. The unbound fraction eluting between 4 and 18 ml as detected by absorption at 230 and 280 nm, was pooled and lyophilized, and the ion-exchange chromatography step was repeated with fresh resin. The unbound peptide fraction was applied in a small volume to a column of Biogel P6 in 100 mM NH4HCO3. Column effluent was monitored by absorption at 230 and 280 nm and by paper electrophoresis of aeid-hydrolyzed aliquots. The peptide-containing effluent was lyophilized, dissolved in formic acid (pH 2.6) and chromatographed on a 0.9 × 15 cm column of AG50 resin, H + form, equilibrated in distilled water. RESULTS

Rhodopsin and its tryptic glycopeptide lacks a free amino terminus When the tryptic peptides of aminoethyl-rhodopsin are subjected to cationexchange chromatography at low pH, a glycopeptide is detected in the unbound peptide fraction eluting from the column (Fig. 1, inset). This material was further purified by gel filtration (Fig. 1). Pool A contains the glycopeptide slightly contaminated with the peptide from pool B. Later fractions (pool C) contain a peptide lacking aromatic amino acids which is the carboxyl-terminal peptide of rhodopsin (Fong, S. L., and Hargrave, P. A., unpublished). After the material from pool A is submitted to cation-exchange chromatography at low pH and low ionic strength, the glycopeptide T1 is obtained in good yield and in pure form (Table I). It is homogeneous by electrophoresis at pH 1.7 and 3.5. Peptide T1 shows no free aminoterminal amino acid by dansylation or Edman degradation. Dansylation of rhodopsin prior to or following the Edman reaction detects no amino-terminal amino acid for the protein. Thus, the amino-terminal blocked peptide T1 is a likely candidate for the amino-terminal peptide of rhodopsin. To gain further evidence for the amino-terminal location of this peptide in rhodopsin we have isolated it as part of a larger tryptic peptide using a method designed to be selective for the preparation of tryptic amino-terminal peptides (based on the procedure of Zabin and Fowler [30]).

88 P6

~.O2. 1.

1.5-

l b 2"0 3"o

E O ~ 1.0--

.5.

(5

80

100

FRACTION

120

140

I

1(30

NUMBER

Fig. 1. Chromatography of the soluble tryptic peptides of aminoethyl-rhodopsin. Inset: Soluble peptides from a tryptic digest of aminoethyl-rhodopsin were chromatographed on a 0.9 × 60 cm column of AG50 resin in 50 mM pyridine acetate buffer (pH 2.4) at 60 ml/min. 3.0-ml fractions were collected. Column fractions which were found to contain glucosamine by amino acid analysis were pooled as indicated. Figure: Pooled glucosamine-containing fractions from chromatography on AG50 (inset) were concentrated and applied to a 1.0 × 200 cm column of Biogel P6 (100-200 mesh) equilibrated in 5 ~ acetic acid. 1.0-ml fractions were collected and their A2a0 ,,1 determined. Peptides were detected by paper electrophoresis of hydrolyzed aliquots. No peptide material was found in the A280 nm-absorbing peaks following pool C. TABLE 1 A M I N O ACID ANALYSIS OF RHODOPSIN PEPTIDES Amino acid

TI a,~

Asx Thr Ser Glx Pro Gly Val Met Tyr Phe Lys GlcN h Yield ( ~)"

3.0 1.0 0.99 1.1 2.1 2.0 0.95 0.28 0.90 1.9 0.89 3.5 35 k

(3) (l) (1) (1) (2) (2) (1) (ly (1) (2) (1)

T1 '"'i

T1-CI"'d

T1-C2

3.0 (3) 2.0 (2) 1.1 (1) 1.3 (1) N.Q. (2) 2.8 (3) 3.1 (3) 0.60 (1) N.Q.(1) j 1.7 (2) 1.1 (1) 1.9 23 k

2.0 (2) 0.97(1) 0.10 (0) 1.1 (I) 1.2 (1) 2.0 (2) 0.20 (0) 0.14 (1) r 0.92 (I) 1.1 (1)

1.0 (1)

1.4 100 j

T1-C3

T1-C1 SI

TI-C1 S2

2.0 (2)

1.o (1) 0.87 (1) 0.97 (1)

1.2 (1) 1.0 (1) 2.0 (2)

1.0 (1)

0.65 (1)

0.86 (1) /.0 (I)

0.91 (1) 0.95 (1) 1.9 721

601

1.6 52 m

40 m

Numbers which are in italics are values used for normalization. Numbers in parentheses are integral values for the tool of amino acid present per tool of peptide. Amino acids present in amounts less than 0.10 residue are not listed. N.Q., Not quantitated but present. a Acid hydrolyzed in a sealed vial in presence of 2 % phenol, bPrepared by paper electrophoresis from papain digestion. Mobility P2 = 52 cm (P3 = 57.5 cm, P5 = 45 cm on the same paper), cPrepared by paper electrophoresis pool B, Fig. 3. Mobility P3 = 62 cm, P4 = 52 cm, P5 = 49 cm, P6 = 40 cm. dAnalyzed by Durrum amino acid analyzer. =Met = 0.88 residues when hydrolyzed in absence of phenol, fMet = 0.76 residues when hydrolyzed in absence of phenol. =Sum of homoserine and homoserine lactone, hall values of glucosamine are low due to the use of conditions for protein hydrolysis. Determined on the short column of the Beckman amino acid analyzer. ~Hydrolyzed 72 h. Jfould not be quantitated due to coelution with glucosamine in this analysis. Detected by paper electrophoresis, kyield based on rhodopsin. ~Yield based on TI. mYield based on T1-CI. "Percent yields do not take into account aliquots removed for analytical purposes during purification.

89 The tryptic glycopeptide is amino terminus o f rhodopsin Succinyl-aminoethyl-rhodopsin was digested with trypsin and carboxypeptidase B, and the digest submitted to cation-exchange chromatography at low pH. The ion-exchange column effluent was further examined by gel filtration (Fig. 2). Peak A was the only portion of eluate which contained peptide material. The bulk of peak A was composed of a peptide which contained tyrosine and glucosamine (and other amino acids) in its composition. The leading edge of peak A contained a minor component distinctive by its content of aminoethylcysteine, alanine and leucine (amino acids which are not present in the major peptide). When the pooled peak A material was subsequently rechromatographed on cation-exchange resin at low ionic strength, the glycopeptide emerged unretarded and in pure form (peptide T I ' , Table I). Peptide T I ' showed no free amino terminal by dansylation, as expected for an aminoterminal peptide prepared from a succinylated protein. Its amino acid composition appears to contain the 16 amino acids of peptide T1, and an additional 2 mol valine and 1 mol each of glycine and threonine.

There are two sites o f carbohydrate attachment in T1 T1 was submitted to digestion by chymotrypsin and three peptides (TI-C1 to T1-C3) were produced. T1-C1 was obtained pure as the unretarded 280 nm absorbing peak from cation-exchange chromatography (by analogy to the purification of T1). The two remaining peptides were obtained by eluting with ammonia from the ion-

T1-CB1 a.a

T1.CB2a

3.0 (3) 0.99 (1) 1.o (1) 1.2 (1) 2.1 (2) 2.1 (2) 0.97 (1)

T1-C1 P1

TI-C1 P2 ~

1.0 (1)

T1-C1 P3 ¢

T1-C1 P4 c

T1-C1 P5 c

T1-C1 P6 c

1.0 (1)

l.O (1)

0.94 (1) 0.80 (1)

1.0 (I) 0.68 (1)

0.93 (1) 0.79 (!)

1.l (1) 1.3 (1)

1.0 (1)

1.0 (1)

8m

33m

6m

28 m

1.0 (1)

TI-C1 P7

1.0 (1) 1.2 (1) 0.70 (1)

0.93 (1) 0.94 (1)

1.2 (1) 1.0 (1)

1.0 (l) g

0.83 (1)

0.90 (1) 1.9 (2) 1.0 (1)

2.4 361

~ 301

1.5 59m

34m

67m

90

1.0E c

E c

i

i

o

4O

60

FRACTION

i

80

1 O0

NUMBER

Fig. 2. Chromatography of the amino-terminal tryptic peptic of succinyl-aminoethyl-rhodopsin. The effluent from AG50 chromatography of the trypsin- and carboxypeptidase B-digested succinylaminoethyl-rhodopsin was chromatographed on a 1.0 × 210 cm column of Biogel P6 equilibrated in 100 mM NH4HCO3. 1.2-ml fractions were collected. Aliquots of fractions were acid hydrolyzed and analyzed for amino acids and hexosamines by high voltage paper electrophoresis. Peak A was pooled as shown. Solid line indicates A230 dotted line A2so.,.. am,

exchange column and separation by gel filtration (Biogel P6 in 5~o acetic acid; (T1-C2, Ks ~ 0.25 and T1-C3, Kd ~ 0.85). T1-C2 contains lysine (Table I) and is, therefore, the carboxyl-terminal region of T1. The sequences Ser-Asx-Lys and ValPro-Phe for T1-C2 and T1-C3, respectively, were determined by the dansyl-Edman method. The TI-C1 is derived from the amino terminus of T1 since no sequence was obtained for it by application of the dansyl-Edman procedure. The compositional data of the three chymotryptic peptides of T1 indicate two sites of attachment of glucosamine; T1-C1 and T 1-C2. The sites of carbohydrate attachment are established by further sequence analysis. The primary sequence o f T1

Peptide T1-C1 was digested with subtilisin and the digest separated by gel filtration. Two peptides were isolated (TI-C1-S1, T1-C1-S2) which accounts for the composition of T1-C1 (Table I). T1-C1-S2 gave the sequence Phe-Tyr, placing it at the carboxyl-end of the parent chymotryptic peptide. T1-C1-S1 gave no sequence by dansyl-Edman, consistent with an amino-terminal position in T1-C1. The papain digest of T1-C1 was also submitted to gel filtration (Fig. 3). Peptides in pool A (T1-C1-P1 and T1-C1-P2) were resolved by ion-exchange chromatography. Peptides in pool B (TI-C1-P3 to T1-C1-P6) were prepared by paper electrophoresis. Pool C contained T1-C1-P7, free tyrosine (Table I). T1-C1-P1 did not degrade by dansyl-Edman and was placed at the amino terminus of T1-C1. T1-C1-P6 was placed at its carboxyl terminus by composition, and TI-C1-P2 in the interior. These three peptides account for the composition of the parent peptide; the other papain peptides are easily accommodated (Fig. 4). The low-yield peptide

91

.6-

.2-

O'.tj

40

60

B0

100

FRACTION

120

140

NUMBER

Fig. 3. Chromatography of the papain digest of peptide T1-C1. 900 nmol of papain-digested peptide T1-C1 was applied to a 1.0 × 220 cm column of Biogel P6 (100-200 mesh) equilibrated in 100 mM NH4HCO3. Fractions of 1.2 ml were collected and their A225.m determined. Aliquots (20%) of alternate tubes were removed for acid hydrolysis and amino acid analysis by high voltage paper electrophoresis. Fractions were pooled as indicated. Peptides in pool A were separated by ion-exchange chromatography [32] and peptides in pool B were separated by preparative paper electrophoresis (see Materials and Methods). T1 CHO x

-

Met

-

Ash

CHO -

Gly

- Thr

-

Olu

-

Gly

-

Pro

~ Ash

Phe

- Tyr

TI-Cl

IA I--

-

Pro,*

T1 -C3

Phe

-

Ser

-,,~

-

Ash

T1-C2

-

Lys

~_,,

TI-CB1

T1-Cl-$1

,,= TI-C1-P1

- Vab

~,,~

IZ~-CB2,_

~ I -

-

-- I A --I~

I T1-C1-$2

TI-C1-P2

I

T1-C1-P6

T1-C1-P5

TI-C1-P7

TI-C1-P3

Fig. 4. Summary of the evidence for the sequence of rhodopsin in peptide TI. The symbol --~ means that the amino acid sequence in that peptide has been determined by the dansyl-Edman technique. (x = amino-terminal blocking group; CHO = carbohydrate). T 1 - C I - P 4 is a mixture as shown by detection o f both phenylalanine and tyrosine as amino terminals and b o t h tyrosine and valine as amino terminals following one r o u n d o f E d m a n degradation. Appearance o f peptides with their composition in a digest of T1-C1 is explained if T1-C1 contained a small p r o p o r t i o n o f molecules with T1-C3 still attached due to incomplete chymotryptic digestion (note the 0.20 m o l valine content o f T 1-C 1 (Table I)). T 1-C l-P4 appears to represent overlapping peptides from residues 9-13 in T I . The cyanogen bromide digest o f peptide T1 yielded two peptides, T1-CB1 and T1-CB2 which account for the composition of T I . T1-CB2 contained only homoserine and its lactone, but the amino acid was ninhydrin negative prior to acid hydrolysis. This places an N-terminal blocked methionine at the amino terminus of T1. The nature o f the blocking g r o u p is currently under investigation.

92 5

The sequence of peptide TI is shown in Fig. 4. The amide status of Glu and Asn were determined from the electrophoretic mobility of T1-CI-P2 (acidic) and 2 T1-C1-P3 (neutral), respectively. The attachment of glucosamine to Asn was inferred from the absence of glucosamine from TI-CB2 and its presence in T1-C1-PI. The 8

15

attachment of glucosamine to Asn is inferred from the digestion of T1-C2 with aminopeptidase M. When the digestion mixture was chromatographed on Biogel P6, the digested peptide coeluted with undigested T1-C2 since the carbohydrate portion of these peptides largely determines their hydrodynamic properties. The glycopeptide fraction gave the amino acid analysis Asxl.0Lysl.0Sero.36GlcN1.8 (78% yield). Comparing with the T1-C2 analysis (Table I) it is clear that there has been extensive removal of serine without a corresponding loss of glucosamine. DISCUSSION The results confirm that the amino terminus of rhodopsin is blocked [5, 15]. A 16-amino acid amino-terminal blocked peptide has been prepared from a tryptic digest of rhodopsin, and its sequence determined. It has a very hydrophilic composition and probably represents a region of this intrinsic membrane protein which is exposed to an aqueous environment on the rod outer segment disc membrane surface. The first nine amino acids contain the sequence of a peptic glycopeptide previously reported by Heller and Lawrence [13]. The amino acid sequence determined by our investigation is the same. However, we find T1 and all peptides derived from its amino terminus to be unreactive in the Edman degradation, in contrast to the previous report [13]. Since the amino terminus of both rhodopsin and peptide T1 is blocked, this peptide is the most reasonable candidate for the amino-terminal region of the protein. The amino-terminal location of the tryptic glycopeptide is further substantiated since its composition is present in a larger peptide isolated by a method specific for preparing such peptides. When a protein is succinylated and digested with trypsin, peptide bond cleavage will be restricted to arginine residues. Following carboxypeptidase B digestion and lowering of the pH sufficiently below the pK of all carboxyl groups, the amino-terminal peptide will be neutral and all other peptides will have a net positive charge. Cation-exchange chromatography under our conditions of low ionic strength will cause all peptides except the amino-terminal peptide to be bound to the resin, allowing only the amino-terminal peptide to pass through. Peptides with pyroglutamic acid amino-terminals may be artifactually generated from an internal glutamine during some conditions of enzymatic digestion, and such peptides would also be prepared by this method. However, peptide TI' is the only peptide isolated from rhodopsin by this method; and structural analysis of peptide T1 shows its amino terminus to be methionine. That peptide T1 is a component of the larger aminoterminal peptide TI' is suggested by the fact that the 16 amino acids of T1 are found in the composition of the 20 amino acid TI'. The difference of Thrl, Glyl, Val2, must 16

21

represent a carboxyl-terminal extension from Lys to the tryptic cleavage site Arg. Additional confirmation comes from the sequence of two thermolytic peptides and one subtilisin peptide of rhodopsin which overlaps them, which shows this sequence

93 17

21

to be Thr-Gly-Val-Val-Arg (Hargrave, P. A., Barber, C. V., Siemens, R. W., Woodin, K. A. and Dreyer, W. J., unpublished). Characterization of the tryptie peptide T1 led to the unexpected finding of a 15

second site of carbohydrate attachment at Asn. Carbohydrate attachment to a single asparagine had been reported and was calculated to account for the entire carbohydrate content of rhodopsin [13]. The sequence analysis shows that this previously 2

described site of oligosaccharide attachment is Asn in the primary sequence of rhodopsin. Although our acid hydrolyses have not been performed under conditions to maximize recovery of glucosamine, it is clear that the glucosamine content of T1 is approximately equally distributed among its chymotryptic cleavage products T1-C1 and T1-C2. The complete carbohydrate analysis of rhodopsin has been reinvestigated recently, and is reported to be mannosegglucosamine5 [7]. The carbohydrate analysis performed on our isolated peptide T1 is consistent with a mannose3_4glucosamine2 unit for each attachment site (Fukuda, M., Papermaster, D. S. and Hargrave, P. A., unpublished). Further investigation will be required to determine whether these two carbohydrate attachment sites account for all of the carbohydrate of rhodopsin. The report of a single concanavalin A binding site per rhodopsin in rod cell membranes [31] is less readily understood in light of our finding of a second site of carbohydrate attachment. ACKNOWLEDGMENTS

I would like to thank Professor William Dreyer (California Institute of Technology) in whose laboratory I first begin to study rhodopsin. I would also like to express my appreciation to Ms. Donna R. Dempsey for her capable technical assistance, Ms. Pat Tindall (Southern Illinois University) and Dr. Paul Fletcher (Yale University) for performing amino acid analysis, and Dr. David Papermaster (Yale University) for his interest in and support of these studies. This work was supported by N.I.H. grants EY 875, EY 1275, GM 21714, and grant G-511 from Fight-for-Sight, Inc., New York, N.Y. 10019. REFERENCES 1 2 3 4 5 6 7 8 9

Daemen, F. J. M., de Grip, W. J. and Jansen, P. A. A. (1972) Biochim Biophys. Acta 271,419-428 Dreyer, W. J., Papermaster, D. S. and Kfihn, H. (1972) Ann. N.Y. Acad. Sci. 195, 61-74 Heitzmann, H. (1972) Nat. New Biol. 235, 114 Robinson, W. E., Gordon-Walker, A. and Bownds, D. (1972) Nat. New Biol. 235, 112-114 Heller, J. (1968) Biochemistry 7, 2906-2913 Schichi, H., Lewis, M. S., Irreverre, F. and Stone, A. L. (1969) J. Biol. Chem. 244, 529-536 Plantner, J. J. and Kean, E. L. (1976) J. Biol. Chem. 251, 1548-1552 Hargrave, P. A. (1976) Vision Res. 16, 1013-1014 Shields, J. E., Dinovo, E. C., Henriksen, R. A., Kimbel, Jr., R. L. and Millar, P. G. (1967) Biochem. Biophys. Acta 147, 238-251 10 Hubbard, R. (1954) J. Gen. Physiol. 37, 381-399 11 Lewis, M. S., Krieg, L. C. and Kirk, W. D. (1974) Exp. Eye Res. 18, 29-40 12 Papermaster, D. S. and Dreyer, W. D. (1974) Biochemistry 13, 2438-2444 13 Heller, J. and Lawrence, M. A. (1970) Biochemistry 9, 864-869 14 Bownds, D. (1967) Nature 216, 1178-1181 15 Albrecht, G. (1957) J. Biol. Chem. 229, 477-487

94 16 Hong, K. and Hubbell, W. L. (1973) Biochemistry 12, 4517-4523 17 Schroeder, D. D. and Shaw, E. (1968) J. Biol. Chem. 243, 2943-2949 18 Dreyer, W. J. and Bynum, E. (1967) in Methods in Enzymology (Hirs, C. H. W., ed.), Vol. l 1, pp. 32-39, Academic Press, New York 19 Schroeder, W. A. (1967) in Methods in Enzymology (Hirs, C. H. W., ed.), Vol. 11, pp. 351-361, Academic Press, New York 20 Lin, Y. C. (1972) Biochim. Biophys. Acta 263, 680--682 21 Cole, R. D. (1967) in Methods in Enzymology (Hirs, C. H. W., ed.), Vol. 11, pp. 315-317, Academic Press, New York 22 Schroeder, W. A., Shelton, J. R0 and Robberson, B. (1967) Biochim. Biophys. Acta 147, 590-592 23 Gray, W. R. (1972) in Methods in Enzymology (Hirs, C. H. W. and Timasheff, S. N., eds.), Vol. 25B, pp. 333-344, Academic Press, New York 24 Bennett, J. C. (1967) in Methods in Enzymology (Hirs, C. H. W., ed.), Vol. 11, pp. 330-339, Academic Press, New York 25 Gray, W. R. and Smith, J. F. (1970) Anal. Biochem. 33, 36-42 26 Hartley, B. S. (1970) Biochem. J. 119, 805-822 27 Mendez, E. and Lai, C. Y. (1975) Anal. Biochem. 65, 281 28 Brockelhurst, K., Carlsson, J., Kierstan, M. P. J. and Crook, E. M. (1973) Biochem. J. 133, 573-584 29 Steers, Jr., E., Craven, G. R., Anfinsen, C. B. and Bethune, J. L. (1965) J. Biol. Chem. 240, 2478-2484 30 Zabin, I. and Fowler, A. V. (1972) J. Biol. Chem. 247, 5432-5435 31 Steinemann, A. and Stryer, L. (1973) Biochemistry 12, 1499-1502 32 Bradshaw, R. A., Garner, W. H. and Gurd, F. R. N. (1969) J. Biol. Chem. 244, 2149-2158