[20]
INTERSTITIAL RETINOL-BINDING PROTEIN
207
[20] Interstitial Retinol-Binding Protein: Purification, C h a r a c t e r i z a t i o n , M o l e c u l a r Cloning, a n d S e q u e n c e B y S H A O - L I N G F O N G a n d C . D . B. BRIDGES
Introduction
Interstitial or interphotoreceptor retinol-binding protein (IRBP) is a large, elongated glycoprotein that is synthesized and secreted by the photoreceptor cells. ~-1° The average molecular weight in vertebrates is 134,200, except in teleosts, where it is about one-half of this value. 11IRBP is found in the interphotoreceptor matrix of the eye, 5:2 where its major function is believed to be the transport of 1l-cis- and all-trans-retinoids between the neural retina and retinal pigment epithelium.13 We have purified and characterized human IRBP, localized its gene to chromosome 10, and obtained its complete cDNA sequence. 4,14,15 The translated human cDNA sequence has been aligned with the amino acid sequences of tryptic peptides from bovine IRBP) 5 The first human IRBP clone (H.4 IRBP) was obtained by screening a 1 A. J. Adler, C. D. Evans, and W. F. Stafford III, J. Biol. Chem. 260, 4850 (1985). 2 A. J. Adler, W. F. Stafford III, and H. S. Slayter, J. Biol. Chem. 262, 13198 (1987). 3 S.-L. Fong, G. I. Liou, R. A. Landers, R. A. Alvarez, and C. D. B. Bridges, J. Biol. Chem. 259, 6534 (1984). 4 S.-L. Fong, G. I. Liou, R. A. Landers, R. A. Alvarez, F. Gonzalez-Femandez, P. A. Glazebrook, D. M. K. Lam, and C. D. B. Bridges, J. Neurochem. 42, 1667 (1984). 5 F. Gonzalez-Fernandez, R. A. Landers, P. A. Glazebrook, S.-L. Fong, G. I. Liou, D. M. K. Lam, and C. D. B. Bridges, J. Cell Biol. 99, 2092 (1984). 6 T. M. Redmond, B. Wiggert, F. A. Robey, N. Y. Nguyen, M. S. Lewis, L. Lee, and G. J. Chader, Biochemistry 24, 787 (1985). 7 j. C. Saari, D. C. Teller, J. W. Crabb, and L. Bredberg, J. Biol. Chem. 260, 195 (1985). s G. 1. Liou, C. D. B. Bridges, S.-L. Fong, R. A. Alvarez, and F. Gonzalez-Fernandez, Vision Res. 22, 1457 (1982). 9 L. Carter-Dawson, R. A. Alvarez, S.-L. Fong, G. I. Liou, H. G. Sperling, and C. D. B. Bridges, Dev. Biol. 116, 431 (1986). 10 T. van Veen, A. Katial, T. Shinohara, D, J. Barrett, B. Wiggert, G. J. Chader, and J. M. Nickerson, FEBS Lett. 208, 133 (1986). ii C. D. B. Bridges, G. I. Liou, R. A. Alvarez, R. A. Landers, A. M. Landry, Jr., and S.-L. Fong, J. Exp. Zool. 239, 335 (1986). 12 A. Bunt-Milam and J. Saari, J. Cell. Biol. 97, 703 (1983). 13 Z.-S. Lin, S.-L. Fong, and C. D. B. Bridges, Vision Res. 29, 1699 (1989). 14 G. I. Liou, S.-L. Fong, J. Gosden, P. van Tuinen, D. H. Ledbetter, S. Christie, D. Rout, S. Bhattacharya, R. G. Cook, Y. Li, C. Wang, and C. D. B. Bridges, Somatic Cell Mol. Genet. 13, 315 (1987). 15 S.-L. Fong and C. D. B. Bridges, J. Biol. Chem. 263, 15330 (1988).
METHODS IN ENZYMOLOGY, VOL. 189
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
208
RECEPTORS, TRANSPORT, A N D BINDING PROTEINS
[20] GGCGGAAGGC AGCTGCACA
-7
GAGCAGGGCCA~GGCTTC~ACA~AGTCCAGGGAGCTTTTGTGCAGGAGCCAGGCCTCC~CCTGGTCCCCATG~AGAGAATG~GTTCTGCTCATGTCCGTGCTGCT~TGTGGCCTC~CT M ~ E W v L L [4 S V L L C G L
48I
GGC C C CACACACCTGTTCCAGC C A A G C C T C ~ A ~ A T G G P T H L F g P $ L V L D
i(5 40
M
G C CAAGGTCCTCTTGGATAACTACTGCTTC C CGGAGAACCTGCTGGGCATGC AGGAAOC CATCCAGCAGGC C A ~ G A K V L L D N Y C ~ P E N L L G M Q E A I Q Q A I K
A~C~ATGA~AT~GA~ATCTCAGA~CCA~AcGcT~G~A~A~AGCcG3G~T~CAGAGCTCcCTGAA~GATcCTCG~`~CTC'GT~ATCTcCTA~A~CCAGcA~C~GAG S H Z I L $ I S D P g T L A S V L T A G V Q S S L N D P R L v I S ¥ E P S O" H x
CCTCC CCCACAAGTCCCAGCACTCACCAGCC T C T C A G A A G A G G A A C T G C T T G C C T ~ C A A J ~ C T C C G C P P P ~) v P A L T S L S E E E L L X w L O R G L A R GTC C C C ~ C A G G A G G T G C T G A G C A T G A T C ~ G T T C C T ~ T G G C C C A C G T ~ A A T C T C A T C ~ C V P G 0 E V L $ g M G E F L V A H V W O ~4 L TCT~TTCC S G I
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AC CTCCGC CTTAGTC-CTGGATCTCC ~ C A ~ C A C A ~ A ~ C A ~ T S A L V L D L R H C T G G
G
CTACATCATCTCCTACCTGCACCCAGGGAACACCATCC T G C A C G T ~ A C A C T A T C T A C A A C C G C C CCTC C A A C A C C A C C A C ~ A ~ C Y I I S Y L ~ P O g T ~ L H V D T I Y N R P S H T T T E I V S V S v O
GGAGA~ACGGT~GACAAGGATGT~ G E R Y G A D K D
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T
CG&QQACATCGCGCACATCCTT~A~C E D Z A H Z [, K Q M Y
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64 20
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GAG E
76 24
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C C L P
5?; ~ H
2S E
x 32
AAGGACTACTACACGCT~CC~CCACCCT~AGCACTTGGCCAGCATGGACTTCTCCAC K O Y Y T L V D R V P T L L (~ H L A S g 0 E A S A
G F L
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T V
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T C T ~ C ~ L V T K L
C ~ A G N A G L
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E CCATATGTCCT~CA~AGCCOCTACAGGACACGGAGCAC P Y V L R ~ V w E P L Q D T !
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CTCATCATC~AC CTGC GC CACAACCCTGGAGGGCC ATC C T ~ C C ~ C L I H D L R H N P G G P S S A V P L
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16 52
G V
TATCT~CCAGC CAC CGCAC CGCCACGGCCGCGGAGGAGTTC GCCTTCCTTATGCAGTCGCTGGGCTGGGCCACACTGGTAGGTGAGATCACCGCGGGCAAC C T ~ C A ~ C Y L L T $ H R T A T A A E E F A F L M Q S L O W A T L v G £ I T A G N L L ACGGT~CGCTC~ACACAC T V P L L D T
C CGAAGGCAGCCTCGCGCTCAC CGTGCCGGTC CTCACCTTC ATC GACAATCACGGCGAGGCC T G G ~ G ~ A G ~ C C P E G $ L A L T V P v L T F I D N H G E A W L G G G V V
P
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G G G C A G A C C A G T ~ C C C T C C T ~ C G G G C C A A G C T ~ C C A G G G C G C C T A C CGCACAGCTGTGGACTTGGAGTCTCTGGCC T C T C A G C T C A C ~ A C ~ C ~ A ~ A C C A C G ~ T S A ~, L R A K ~ A ~ G A Y R T A v D L E S L A S Q L T A D L ~ E V S G D G C~AGTGTTCCACAGC R 5 L V F H
S
CC T G G C G A G C T ~ T A G A G G A A G C A C P G E ~ V V E E A M
(TI~,CI~CC C C ~ G C C ~ A C C T G C G ~ ' F r G A C
CCCCACCAC C C C C T C ~ T C C C CT C T C C A G A G G A G ~ A C C T A C ~ A ~ A ~ C ~ G ~ G A C A G A G P P P P P A V P S P E E L T ¥ L I E A L F K T
A
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A ~ I ' G A A C - G C C ~ C A C A ~ C ~ A ~ C
A G ~ A C A ¢ ~ C G
23
~TCGACC'.~q~GCTA~AA~CcTC~&g~TACTC~AC~G~CAT~cGCTGCT~TGCTCCTA~TTCT.FrGA~GCAGAGC~CCG~CAGCACCTGTA~A~A~cCACC r., v r O L R Y N P G S Y $ T A I P L L C $ Y F F E A E P R Q H L Y S V F D R A T
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C T C ~ C G A G G A G G C C C T G G A C A A A G C C C A G G A A G T G C T G G A G T T CC A C C A A A G C C T G G G G G C C T T ~ G G G C A C A G G G C A C CTGCT~CC CA~A~C~CAGA~TC L A £ E A L O K A Q E V L £ ~ H Q S L G A L V E G T G H L L £ A H Y A R P E x £ R
24 80
V
TC~CGGA~"~'G"~W~AC S K V T E V W
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c'~rGc C CCAGGTCGCCGGC C AGC GCTACGGCTCACACAAGGAC CTCTACATCL"rGA'I'GA~ CACAC C A L E ~ v A G ~ g Y G S H K D L Y I L M $ H T S H T
AC CATGCAGG ACCTGCAGCGGGC CACGGTCATTGGGGAGCCCACGGCCGGAGGCGCA C T C T C T ~ A T C T A C C A ~ T G G G C A G C A G C C C T M Q D L Q R A T v I G E P T A G G A L S V G I Y ~ V G S S ' I
P A
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G A A G T ~ C C T A G C C G A G A T C CTGGGGGCTGACCTGCAGATGCTCTCCGGAGACCCACACCTGAAGG CAGCCCATATCCCTGAGAATGCC ~ A C CGC A ~ C ~ E V A L A E I L G A D L Q M L S G D P H L K A A H I P E N A K D R I P
C G
A
CAC C A A A C T G A G C ~ C T K L S G L
L
AGA~C~A~ 0 S R Y
T
GGCC GGGAAGCTGGTGGCTGATAACTATGCCTCTCC C G A G C T ~ C A A G A T G G C A G K L V A D N Y A S A E L G A K M p V
c GA~ ~ACAC E A F A
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~ A T A T G C ATC C A ~ C C CACCCAGATG L Y A $ M P T 0 M
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CC C A C G G T ~ A G A C P T V L Q
14 48
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G~Cc~cGGC~CGTC~AcCTCTT~A~ACCT&TGATcGCCG~A~AACATCAcG~AC~AG~ACTTcAGC~ACATGGAG~TC~CGGGC~AcGCTA~AGcAC~AA~GTGGGGTG
G S
11 36
25 84 26 88
K
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28 92
CA~AC~CA R v T
s
29 9~
ATG M
]G IG
g
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C C I V
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[20]
209
INTERSTITIAL RETINOL-BINDING PROTEIN
CAGATCCCTTCCCCTGAAGTATTI~AAGAGCTGATCAAGTTTTCCTTCCA C A C T A A C ~ A G G A C A A C A T T C ~ G C T A C T T G A G ~ E T I ~ A C A ~ A C G G T G A C d Y I ~ ' T ~ C A C Q I P $ P £ V g E E L I K F $ F 1~ T M V L E D N I G Y L R F D M F G D G E L L X
T
3168 1040
CAGGTCTCCAC'GCTC~ITd~TGGAGCACATCTCsGAAGAAGATCATGCACAC~ATGCC A T ~ A T C A T C G A C A T G A C ' G T T C A A C A T C ~ C C C AC A T C ~ C A ~ C C A T ~ C C Q V S R L L V E H I W K K I M H T D A H I I D M R F N I G G P T S $ Z P ~ ~ C $
]288 1080
TACTTCTTTGATGAAGGCC CTC C AG'£TC T G ~ A C A A G A T C T A C A G C C GGC C T G A T G A C T C T G T C A G T G A A C T ~ AC AC AC GC C C A G G T T G T A G G ~ C G ~ A ~ Y F g D E G P P V L L P K I V S R P D D $ V S E L W T H A ~ V V G E R Y
3408 1120
G
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AGCATC~TCATTCTGACCAGC AGTGTG ACGGC CGGC AC C G C GGAGGAGTTCAC CTATATC ATGAAGAGG CTGGGC CGGGC C C T G G T C A T T G G G G A ~ A C CAGT G ~ C ~ C $ M V I L T S S V T A G T A E g F T Y I M K R L G R A L V I G g V T $ G G
C
C CACAGACCTACCACGTGGATGACACC AAC CT CTACCTCACTATC C C C AC GGC C CGTTCTGTGGCIGGCCTCGGATGGCAGCTCCTGGGAAGG G G ~ T G A C P Q T Y H V D D T N L Y L T Z P T A R $ V ~ A S D G S S W g G V G V D A C CTGCAGAAGAC~CTCTCGCCAGGGCCAAGGAGATGCTCCAGCAC AAC CAGCTGAGGGTGAAGCGGAGC C CAGGCCTGCAGGACC AC C T ~ A L A R A K g M L ~ H N ~ L R V K a $ P G L Q D H L
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3528 i~60 3648 1200 3768
F
Q G C A G A C A G A A C C T C T ~ G A C A C A C A CCAAGGGC ACTC CTGC AGGTGG C C CGGC CTGAGGTTCC CAGG AC CAG CAAAGGGGC C T G C T G A G C T C T ~ T T A G G ~ AC A G C T G G A ~ T A
3888
TATATAcA~ACAcA~ACATGTATATA~A~ATATATAT~TGTATGTATATATAT~TATATATATATGGCT~CCAATAAC~A~CTAAATTTTAA~AAAGGTTCCTTCTAAGTGGTAGAA~T 4008
TC~GGTGGTA x~x-x-~-r ACCTTC CTTCI~ CATAC TTTG C~ ~ ~ ~ ~ L-~'CTTAAATACTCATTAATGTGC ATATAT CATTA'iTTTCAGATQC AG CT AT C ATTATTCC AAAATA CAAAATAAAGAA
GATAAAATAAA
FIG. 1. (continued)
human retina hgtl0 c D N A library with a 258-base-pair bovine probe (B23) obtained from a bovine retina hgtl 1 expression library. H.4 IRBP was then employed as a probe to enable isolation and purification of three overlapping human c D N A clones (H8, H12, and H18) from the same human retina hgtl0 c D N A library. 15 The complete cDNA sequence of 4230 bases (Fig. 1) was derived from the sequences of H.12, H.4, and H.18. Purification of Bovine IRBP Because human eyes are not available in large quantities, 4 most biochemical studies have been carried out on the bovine protein. Bovine IRBP is prepared from 100-200 eyes according to Fong e t a/. 3A6 The interphotoreceptor matrix preparation is dialyzed and loaded on a DEAE16 S.-L. Fong, G. I. Liou, and C. D. B. Bridges, this series, Vol. 123, p. 102. FIG. 1. Nucleotide and deduced amino acid sequences of human IRBP. The aminoterminal sequence obtained for the mature protein is underlined from amino acid residues 1-32 (gaps denote uncertain residue identification). The tryptic peptide sequences of bovine IRBP corresponding to the deduced human IRBP sequence are also underlined; mismatches are indicated below the underline. Uncertain residues identified by a gas-phase protein sequencer or by mismatches between the peptide sequences and the deduced bovine IRBP sequence [D. E. Borst, T. M. Redmond, J. E. Elser, M. A. Gorda, B. Wiggert, G. J. Chader, and J. M. Nickerson, J. Biol. Chem. 264, 1115 (1989)] are indicated by an x below the underline. The translation initiation site and the stop codon (TAG) are overlined.
4128
210
RECEPTORS, TRANSPORT, AND BINDING PROTEINS
[20]
cellulose column. The eluted fractions containing IRBP are pooled and passed through a concanavalin A column, which binds IRBP. Fractions eluted with methyl a-o-mannopyranoside are concentrated in the presence of 1% octyl fl-D-glucopyranoside. Sugar and detergent are removed by a final gel-filtration step on Sepharose CL-4B. Preparation and Characterization of Tryptic Peptides of Bovine IRBP Purified bovine IRBP (23.2 mg) is reduced under a nitrogen barrier with 0.2 g 2-mercaptoethanol in 15 ml of 0.6 M Tris-HC1 (pH 8.6), 6 M guanidine-HCl, and 0.2% EDTA. ~7The sulfhydryl groups of cysteine residues are then modified by stirring for 15 min with 476 mg iodoacetic acid in 2 ml of 1 N NaOH. After dialysis against water, the carboxymethylated IRBP is resuspended in 5 ml of 0.2 N N-ethylmorpholinoacetic acid buffer (pH 8.2). Trypsin digestion is carried out by incubating at 37° with two consecutive additions of 1.5 and 2% (w/w) trypsin solutions at 2-hr intervals. At the end of the incubation, the tryptic peptides are separated on a Sephadex G-50 column (1 × 100 cm) and eluted with 0.1 M NH4HCO3. Six 280-nm absorption peaks (T1 through T6) are pooled as illustrated by the typical chromatograph in Fig. 2A. Each pooled peak is lyophilized and dissolved in 0.1% trifluoroacetic acid. Aliquots of each peak (except T1) are freed of particulate matter by centrifugal filtration (Rainin, Woburn, MA, 0.2-/zm cellulosic membranes) and injected into an FPLC (fast purification liquid chromatography) system based on a Pharmacia reversed-phase Pep RPC HR 5/5 column. Gradient chromatography is carried out with two Altex-Beckman Model 100 pumps, an Altex 400 solvent programmer, and an Altex-Hitachi Model 155-10 variable-wavelength detector. The effluent should be continuously monitored at 280 nm. A typical chromatogram obtained by FPLC of T3 is shown in Fig. 2B. Five to ten percent of each fraction derived from each FPLC peak is loaded on a gas-phase protein sequencer (Applied Biosystems, Foster City, CA) for amino acid sequence determination. In our experience, most of the peaks from the FPLC reversed-phase column contain more than one peptide. However, many of them are present in different relative amounts and can therefore be sequenced directly because the different amounts of phenylthiohydantoin (PTH)-amino acids produced in each cycle of Edman degradation can be used to distinguish different peptides. Thus, peaks R1 and R3 in Fig. 2B are found to contain two peptides each. Peaks R4, R5, and R6 contained single peptides. If the amounts of two or more peptides are about the same, however, further separation on an17 A. M. Crestfield, S. Moore, and W. H. Stein, J. Biol. Chem. 238, 622 (1963).
[20]
INTERSTITIAL RETINOL-BINDING PROTEIN
211
A 0.8
T1
T2
E P
T3
T4
T5
T6
ii
0.7
Vo 17.2 k
0.6
Vi
o
0.5 c
0.4, 0.3!
.Q
0.2. 0.1 0.0
• .
0
.
.
.
.
50
Fraction
.
.
.
.
.
.
.
.
100 number
u
•
•
150
C
OD280
O D280
•~ - r r
I 25
I MIN
d
cD
I 35
I
I 10
MIN
I 15
FIG. 2. (A) Size fractionation of tryptic peptides of bovine IRBP. Tryptic peptides generated from reduced carboxymethylated bovine IRBP were loaded on a Sephadex G-50 column (1 × 100 cm) and eluted with 0.1 N NH4HCO3. Fractions of I ml were collected. Vo, void volume; V~, included volume. (B) Reversed-phase chromatography ofT3 on a Pep RPC HR 5/5 column. Eluent A, 0.1% trifluoroacetic acid in water; eluent B, 0.1% trifluoroacetic acid in acetonitrile. Flow rate: 0.7 ml/min. Linear gradient 0-50% (B in A) in 30 min. (C) Ion-exchange chromatography of R2 on a Mono Q column. Eluent A, 20 mM Tris-HCl, pH 7.8; eluent B, 1 M NaC! in eluent A. Flow rate: 1 ml/min. Concave gradient, 0-50% (B in A) in 15 min, exponent m = 3.
212
RECEPTORS, TRANSPORT, AND BINDING PROTEINS
[20]
TABLE I AMINO ACID SEQUENCES OF 10 BOVINE IRBP TRYPTIC PEPTIDES PURIFIED FROM FRACTION T3
Peptide Rl.l. R1.2b,, '
R2M1 R2M2 R2M3 R3.1 b R3.2b. C R4 b R5 R6 b
Sequence" IYNRPXXSVSELWTLSQLEGER LXXTSALVLXL SVGAADGSSWEGVGVVPDVAVPAEAALTR ALVIGEVTSGGCQPPQTYHVDDTDLYLTIPTAR SLGPLGEGSXTWEGSGVLPCVGTPAEQALEK SQEILSISDPQTLAHVLTAGVX XASTGEXXXLAGVEPDXXVPM XNIGGPTSSiSALCSYFFXEGPPI EYYTLVDRVPALLSHLAAMDLSSVVSEDDLVTK FDSFADASVLEVLGPYILHQVWEP
a X denotes residues not identified during peptide sequencing or mistmatched residues between the peptide sequence and the deduced amino acid sequence for bovine IRBP. b These sequences were not completed to the terminal R or K. ¢ The sequence is not underlined in Fig. 1.
other column is necessary. We have found that Mono Q columns are a good choice for this purpose. The Mono Q column is packed with a hydrophilic polymer containing quaternary amine groups that remain equally charged over a wide pH range of 2-12. When the major peak (R2) from the reversed-phase column is chromatographed on this anion-exchange column, three peptides are easily resolved, namely, R2M1, R2M2, and R2M3. The sequences of 10 tryptic peptides obtained from T3 are shown in Table I. The same strategy may be applied to the other T fractions. The aminoterminal sequence of the protein can be obtained directly from purified bovine IRBP 18,19 (see underlined amino acid residues 1-32 in Fig. 1). During gel-filtration chromatography, T1 elutes in the void volume. This corresponds to a molecular weight of 30,000 or larger, which cannot be analyzed on the Pep RPC column, which has an exclusion limit of 6,000. Molecular Biology In our original work, s a bovine retina eDNA library in the expression vector hgtl 1 provided by Drs. Khorana and Oprian (Massachusetts Insti~8S.-L. Fong, R. G. Cook, R. A. Alvarez, G. I. Liou, and C. D. B. Bridges, F E B S L e t t . 205, 309 (1986). 19 T. M. Redmond, B. Wiggert, F. A. Robey, and G. J. Chader, B i o c h e m . J. 240, 19 (1986).
[ 2 1]
INTERPHOTORECEPTOR R E T I N O I D - B I N D I N G PROTEIN
213
tute of Technology, Cambridge, MA) was screened with polyclonal rabbit anti-bovine IRBP immunoglobulin G (IgG) and ~25I-labeled protein or horseradish peroxidase-labeled goat anti-rabbit IgG (Bio-Rad, Richmond, CA). The procedure used was described by Young and Davis 2°and Huynh et al. 21 After the k phage was absorbed to the YI090 host cell, 105 plaqueforming units (pfu) was plated on a 150-mm plate. IPTG-containing nitrocellulose filters were overlaid on the plate and saturated with 20% fetal calf serum and 5% nonfat milk. Following incubation with anti-bovine IRBP IgG, the filter was treated with either ~25I-labeled protein A or horseradish peroxidase-labeled goat anti-rabbit IgG. When the human retina cDNA hgtl0 library (provided by Dr. Jeremy Nathans, Johns Hopkins School of Medicine, Baltimore, MD) was screened, nick-translated, 32p-labeled cDNA fragments (bovine probe B23 and H.4 IRBP) were used as probes. The procedure of Maniatis et al. 22 was followed. Phage DNA from positive, isolated clones was purified by the plate lysate method using LambdaSorb (Promega Biotec, Madison, WI). The insert was removed from the phage by E c o R I , purified by agarose gel electrophoresis, and subcloned into M13mpl8 and M13mpl9 for sequencing by the chain-termination method23using the procedure of Dale et al. 24 20 R. A. Young and R. W. Davis, Science 222, 778 (1983). 21 T. V. Huynh, R. A. Young, and R. W. Davis, in " D N A Cloning, A Practical Approach" (D. M. Glover, ed.), Vol. 1. IRL Press, Oxford and Washington, D.C., 1985. 22 T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982. 23 F. Sanger, S. Nicklen, and A. R. Coulson, Proc. Natl. Acad. Sci. U.S.A. 74, 5463 (1979). 24 R. M. K. Dale, B. A. McClure, and J. P. Houchins, Plasmid 13, 31 (1985).
[21] P u r i f i c a t i o n a n d A s s a y of I n t e r p h o t o r e c e p t o r Retinoid-Binding Protein from the Eye B y A L I C E J. A D L E R , GERALD J. CHADER, and BARBARA WIGGERT
Introduction Interphotoreceptor retinoid-binding protein (IRBP) is an extracellular carrier of vitamin A and other lipids. 1-3 It occurs primarily in photosensii A. J. Adler and K. J. Martin, Biochem. Biophys. Res. Commun. 108, 1601 (1982). 2 G. I. Liou, C. D. B. Bridges, S.-L. Fong, R. A. Alvarez, and F. Gonzalez-Fernandez, Vision Res. 22, 1457 (1982). 3 y. L. Lai, B. Wiggert, Y. P. Liu, and G. J. Chader, Nature (London) 298, 848 (1982).
METHODS IN ENZYMOLOGY, VOL. 189
Copyright © 1990by Academic Press, Inc. All rights of reproduction in any form reserved.
INTERSTITIAL RETINOL-BINDING PROTEIN
207
[20] Interstitial Retinol-Binding Protein: Purification, C h a r a c t e r i z a t i o n , M o l e c u l a r Cloning, a n d S e q u e n c e B y S H A O - L I N G F O N G a n d C . D . B. BRIDGES
Introduction
Interstitial or interphotoreceptor retinol-binding protein (IRBP) is a large, elongated glycoprotein that is synthesized and secreted by the photoreceptor cells. ~-1° The average molecular weight in vertebrates is 134,200, except in teleosts, where it is about one-half of this value. 11IRBP is found in the interphotoreceptor matrix of the eye, 5:2 where its major function is believed to be the transport of 1l-cis- and all-trans-retinoids between the neural retina and retinal pigment epithelium.13 We have purified and characterized human IRBP, localized its gene to chromosome 10, and obtained its complete cDNA sequence. 4,14,15 The translated human cDNA sequence has been aligned with the amino acid sequences of tryptic peptides from bovine IRBP) 5 The first human IRBP clone (H.4 IRBP) was obtained by screening a 1 A. J. Adler, C. D. Evans, and W. F. Stafford III, J. Biol. Chem. 260, 4850 (1985). 2 A. J. Adler, W. F. Stafford III, and H. S. Slayter, J. Biol. Chem. 262, 13198 (1987). 3 S.-L. Fong, G. I. Liou, R. A. Landers, R. A. Alvarez, and C. D. B. Bridges, J. Biol. Chem. 259, 6534 (1984). 4 S.-L. Fong, G. I. Liou, R. A. Landers, R. A. Alvarez, F. Gonzalez-Femandez, P. A. Glazebrook, D. M. K. Lam, and C. D. B. Bridges, J. Neurochem. 42, 1667 (1984). 5 F. Gonzalez-Fernandez, R. A. Landers, P. A. Glazebrook, S.-L. Fong, G. I. Liou, D. M. K. Lam, and C. D. B. Bridges, J. Cell Biol. 99, 2092 (1984). 6 T. M. Redmond, B. Wiggert, F. A. Robey, N. Y. Nguyen, M. S. Lewis, L. Lee, and G. J. Chader, Biochemistry 24, 787 (1985). 7 j. C. Saari, D. C. Teller, J. W. Crabb, and L. Bredberg, J. Biol. Chem. 260, 195 (1985). s G. 1. Liou, C. D. B. Bridges, S.-L. Fong, R. A. Alvarez, and F. Gonzalez-Fernandez, Vision Res. 22, 1457 (1982). 9 L. Carter-Dawson, R. A. Alvarez, S.-L. Fong, G. I. Liou, H. G. Sperling, and C. D. B. Bridges, Dev. Biol. 116, 431 (1986). 10 T. van Veen, A. Katial, T. Shinohara, D, J. Barrett, B. Wiggert, G. J. Chader, and J. M. Nickerson, FEBS Lett. 208, 133 (1986). ii C. D. B. Bridges, G. I. Liou, R. A. Alvarez, R. A. Landers, A. M. Landry, Jr., and S.-L. Fong, J. Exp. Zool. 239, 335 (1986). 12 A. Bunt-Milam and J. Saari, J. Cell. Biol. 97, 703 (1983). 13 Z.-S. Lin, S.-L. Fong, and C. D. B. Bridges, Vision Res. 29, 1699 (1989). 14 G. I. Liou, S.-L. Fong, J. Gosden, P. van Tuinen, D. H. Ledbetter, S. Christie, D. Rout, S. Bhattacharya, R. G. Cook, Y. Li, C. Wang, and C. D. B. Bridges, Somatic Cell Mol. Genet. 13, 315 (1987). 15 S.-L. Fong and C. D. B. Bridges, J. Biol. Chem. 263, 15330 (1988).
METHODS IN ENZYMOLOGY, VOL. 189
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
208
RECEPTORS, TRANSPORT, A N D BINDING PROTEINS
[20] GGCGGAAGGC AGCTGCACA
-7
GAGCAGGGCCA~GGCTTC~ACA~AGTCCAGGGAGCTTTTGTGCAGGAGCCAGGCCTCC~CCTGGTCCCCATG~AGAGAATG~GTTCTGCTCATGTCCGTGCTGCT~TGTGGCCTC~CT M ~ E W v L L [4 S V L L C G L
48I
GGC C C CACACACCTGTTCCAGC C A A G C C T C ~ A ~ A T G G P T H L F g P $ L V L D
i(5 40
M
G C CAAGGTCCTCTTGGATAACTACTGCTTC C CGGAGAACCTGCTGGGCATGC AGGAAOC CATCCAGCAGGC C A ~ G A K V L L D N Y C ~ P E N L L G M Q E A I Q Q A I K
A~C~ATGA~AT~GA~ATCTCAGA~CCA~AcGcT~G~A~A~AGCcG3G~T~CAGAGCTCcCTGAA~GATcCTCG~`~CTC'GT~ATCTcCTA~A~CCAGcA~C~GAG S H Z I L $ I S D P g T L A S V L T A G V Q S S L N D P R L v I S ¥ E P S O" H x
CCTCC CCCACAAGTCCCAGCACTCACCAGCC T C T C A G A A G A G G A A C T G C T T G C C T ~ C A A J ~ C T C C G C P P P ~) v P A L T S L S E E E L L X w L O R G L A R GTC C C C ~ C A G G A G G T G C T G A G C A T G A T C ~ G T T C C T ~ T G G C C C A C G T ~ A A T C T C A T C ~ C V P G 0 E V L $ g M G E F L V A H V W O ~4 L TCT~TTCC S G I
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AC CTCCGC CTTAGTC-CTGGATCTCC ~ C A ~ C A C A ~ A ~ C A ~ T S A L V L D L R H C T G G
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CTACATCATCTCCTACCTGCACCCAGGGAACACCATCC T G C A C G T ~ A C A C T A T C T A C A A C C G C C CCTC C A A C A C C A C C A C ~ A ~ C Y I I S Y L ~ P O g T ~ L H V D T I Y N R P S H T T T E I V S V S v O
GGAGA~ACGGT~GACAAGGATGT~ G E R Y G A D K D
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CG&QQACATCGCGCACATCCTT~A~C E D Z A H Z [, K Q M Y
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GAG E
76 24
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C C L P
5?; ~ H
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x 32
AAGGACTACTACACGCT~CC~CCACCCT~AGCACTTGGCCAGCATGGACTTCTCCAC K O Y Y T L V D R V P T L L (~ H L A S g 0 E A S A
G F L
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T V
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T C T ~ C ~ L V T K L
C ~ A G N A G L
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E CCATATGTCCT~CA~AGCCOCTACAGGACACGGAGCAC P Y V L R ~ V w E P L Q D T !
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CTCATCATC~AC CTGC GC CACAACCCTGGAGGGCC ATC C T ~ C C ~ C L I H D L R H N P G G P S S A V P L
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16 52
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TATCT~CCAGC CAC CGCAC CGCCACGGCCGCGGAGGAGTTC GCCTTCCTTATGCAGTCGCTGGGCTGGGCCACACTGGTAGGTGAGATCACCGCGGGCAAC C T ~ C A ~ C Y L L T $ H R T A T A A E E F A F L M Q S L O W A T L v G £ I T A G N L L ACGGT~CGCTC~ACACAC T V P L L D T
C CGAAGGCAGCCTCGCGCTCAC CGTGCCGGTC CTCACCTTC ATC GACAATCACGGCGAGGCC T G G ~ G ~ A G ~ C C P E G $ L A L T V P v L T F I D N H G E A W L G G G V V
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G G G C A G A C C A G T ~ C C C T C C T ~ C G G G C C A A G C T ~ C C A G G G C G C C T A C CGCACAGCTGTGGACTTGGAGTCTCTGGCC T C T C A G C T C A C ~ A C ~ C ~ A ~ A C C A C G ~ T S A ~, L R A K ~ A ~ G A Y R T A v D L E S L A S Q L T A D L ~ E V S G D G C~AGTGTTCCACAGC R 5 L V F H
S
CC T G G C G A G C T ~ T A G A G G A A G C A C P G E ~ V V E E A M
(TI~,CI~CC C C ~ G C C ~ A C C T G C G ~ ' F r G A C
CCCCACCAC C C C C T C ~ T C C C CT C T C C A G A G G A G ~ A C C T A C ~ A ~ A ~ C ~ G ~ G A C A G A G P P P P P A V P S P E E L T ¥ L I E A L F K T
A
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A ~ I ' G A A C - G C C ~ C A C A ~ C ~ A ~ C
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23
~TCGACC'.~q~GCTA~AA~CcTC~&g~TACTC~AC~G~CAT~cGCTGCT~TGCTCCTA~TTCT.FrGA~GCAGAGC~CCG~CAGCACCTGTA~A~A~cCACC r., v r O L R Y N P G S Y $ T A I P L L C $ Y F F E A E P R Q H L Y S V F D R A T
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24 80
V
TC~CGGA~"~'G"~W~AC S K V T E V W
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c'~rGc C CCAGGTCGCCGGC C AGC GCTACGGCTCACACAAGGAC CTCTACATCL"rGA'I'GA~ CACAC C A L E ~ v A G ~ g Y G S H K D L Y I L M $ H T S H T
AC CATGCAGG ACCTGCAGCGGGC CACGGTCATTGGGGAGCCCACGGCCGGAGGCGCA C T C T C T ~ A T C T A C C A ~ T G G G C A G C A G C C C T M Q D L Q R A T v I G E P T A G G A L S V G I Y ~ V G S S ' I
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C G
A
CAC C A A A C T G A G C ~ C T K L S G L
L
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T
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c GA~ ~ACAC E A F A
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~ A T A T G C ATC C A ~ C C CACCCAGATG L Y A $ M P T 0 M
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G~Cc~cGGC~CGTC~AcCTCTT~A~ACCT&TGATcGCCG~A~AACATCAcG~AC~AG~ACTTcAGC~ACATGGAG~TC~CGGGC~AcGCTA~AGcAC~AA~GTGGGGTG
G S
11 36
25 84 26 88
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28 92
CA~AC~CA R v T
s
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ATG M
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[20]
209
INTERSTITIAL RETINOL-BINDING PROTEIN
CAGATCCCTTCCCCTGAAGTATTI~AAGAGCTGATCAAGTTTTCCTTCCA C A C T A A C ~ A G G A C A A C A T T C ~ G C T A C T T G A G ~ E T I ~ A C A ~ A C G G T G A C d Y I ~ ' T ~ C A C Q I P $ P £ V g E E L I K F $ F 1~ T M V L E D N I G Y L R F D M F G D G E L L X
T
3168 1040
CAGGTCTCCAC'GCTC~ITd~TGGAGCACATCTCsGAAGAAGATCATGCACAC~ATGCC A T ~ A T C A T C G A C A T G A C ' G T T C A A C A T C ~ C C C AC A T C ~ C A ~ C C A T ~ C C Q V S R L L V E H I W K K I M H T D A H I I D M R F N I G G P T S $ Z P ~ ~ C $
]288 1080
TACTTCTTTGATGAAGGCC CTC C AG'£TC T G ~ A C A A G A T C T A C A G C C GGC C T G A T G A C T C T G T C A G T G A A C T ~ AC AC AC GC C C A G G T T G T A G G ~ C G ~ A ~ Y F g D E G P P V L L P K I V S R P D D $ V S E L W T H A ~ V V G E R Y
3408 1120
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C
C CACAGACCTACCACGTGGATGACACC AAC CT CTACCTCACTATC C C C AC GGC C CGTTCTGTGGCIGGCCTCGGATGGCAGCTCCTGGGAAGG G G ~ T G A C P Q T Y H V D D T N L Y L T Z P T A R $ V ~ A S D G S S W g G V G V D A C CTGCAGAAGAC~CTCTCGCCAGGGCCAAGGAGATGCTCCAGCAC AAC CAGCTGAGGGTGAAGCGGAGC C CAGGCCTGCAGGACC AC C T ~ A L A R A K g M L ~ H N ~ L R V K a $ P G L Q D H L
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3528 i~60 3648 1200 3768
F
Q G C A G A C A G A A C C T C T ~ G A C A C A C A CCAAGGGC ACTC CTGC AGGTGG C C CGGC CTGAGGTTCC CAGG AC CAG CAAAGGGGC C T G C T G A G C T C T ~ T T A G G ~ AC A G C T G G A ~ T A
3888
TATATAcA~ACAcA~ACATGTATATA~A~ATATATAT~TGTATGTATATATAT~TATATATATATGGCT~CCAATAAC~A~CTAAATTTTAA~AAAGGTTCCTTCTAAGTGGTAGAA~T 4008
TC~GGTGGTA x~x-x-~-r ACCTTC CTTCI~ CATAC TTTG C~ ~ ~ ~ ~ L-~'CTTAAATACTCATTAATGTGC ATATAT CATTA'iTTTCAGATQC AG CT AT C ATTATTCC AAAATA CAAAATAAAGAA
GATAAAATAAA
FIG. 1. (continued)
human retina hgtl0 c D N A library with a 258-base-pair bovine probe (B23) obtained from a bovine retina hgtl 1 expression library. H.4 IRBP was then employed as a probe to enable isolation and purification of three overlapping human c D N A clones (H8, H12, and H18) from the same human retina hgtl0 c D N A library. 15 The complete cDNA sequence of 4230 bases (Fig. 1) was derived from the sequences of H.12, H.4, and H.18. Purification of Bovine IRBP Because human eyes are not available in large quantities, 4 most biochemical studies have been carried out on the bovine protein. Bovine IRBP is prepared from 100-200 eyes according to Fong e t a/. 3A6 The interphotoreceptor matrix preparation is dialyzed and loaded on a DEAE16 S.-L. Fong, G. I. Liou, and C. D. B. Bridges, this series, Vol. 123, p. 102. FIG. 1. Nucleotide and deduced amino acid sequences of human IRBP. The aminoterminal sequence obtained for the mature protein is underlined from amino acid residues 1-32 (gaps denote uncertain residue identification). The tryptic peptide sequences of bovine IRBP corresponding to the deduced human IRBP sequence are also underlined; mismatches are indicated below the underline. Uncertain residues identified by a gas-phase protein sequencer or by mismatches between the peptide sequences and the deduced bovine IRBP sequence [D. E. Borst, T. M. Redmond, J. E. Elser, M. A. Gorda, B. Wiggert, G. J. Chader, and J. M. Nickerson, J. Biol. Chem. 264, 1115 (1989)] are indicated by an x below the underline. The translation initiation site and the stop codon (TAG) are overlined.
4128
210
RECEPTORS, TRANSPORT, AND BINDING PROTEINS
[20]
cellulose column. The eluted fractions containing IRBP are pooled and passed through a concanavalin A column, which binds IRBP. Fractions eluted with methyl a-o-mannopyranoside are concentrated in the presence of 1% octyl fl-D-glucopyranoside. Sugar and detergent are removed by a final gel-filtration step on Sepharose CL-4B. Preparation and Characterization of Tryptic Peptides of Bovine IRBP Purified bovine IRBP (23.2 mg) is reduced under a nitrogen barrier with 0.2 g 2-mercaptoethanol in 15 ml of 0.6 M Tris-HC1 (pH 8.6), 6 M guanidine-HCl, and 0.2% EDTA. ~7The sulfhydryl groups of cysteine residues are then modified by stirring for 15 min with 476 mg iodoacetic acid in 2 ml of 1 N NaOH. After dialysis against water, the carboxymethylated IRBP is resuspended in 5 ml of 0.2 N N-ethylmorpholinoacetic acid buffer (pH 8.2). Trypsin digestion is carried out by incubating at 37° with two consecutive additions of 1.5 and 2% (w/w) trypsin solutions at 2-hr intervals. At the end of the incubation, the tryptic peptides are separated on a Sephadex G-50 column (1 × 100 cm) and eluted with 0.1 M NH4HCO3. Six 280-nm absorption peaks (T1 through T6) are pooled as illustrated by the typical chromatograph in Fig. 2A. Each pooled peak is lyophilized and dissolved in 0.1% trifluoroacetic acid. Aliquots of each peak (except T1) are freed of particulate matter by centrifugal filtration (Rainin, Woburn, MA, 0.2-/zm cellulosic membranes) and injected into an FPLC (fast purification liquid chromatography) system based on a Pharmacia reversed-phase Pep RPC HR 5/5 column. Gradient chromatography is carried out with two Altex-Beckman Model 100 pumps, an Altex 400 solvent programmer, and an Altex-Hitachi Model 155-10 variable-wavelength detector. The effluent should be continuously monitored at 280 nm. A typical chromatogram obtained by FPLC of T3 is shown in Fig. 2B. Five to ten percent of each fraction derived from each FPLC peak is loaded on a gas-phase protein sequencer (Applied Biosystems, Foster City, CA) for amino acid sequence determination. In our experience, most of the peaks from the FPLC reversed-phase column contain more than one peptide. However, many of them are present in different relative amounts and can therefore be sequenced directly because the different amounts of phenylthiohydantoin (PTH)-amino acids produced in each cycle of Edman degradation can be used to distinguish different peptides. Thus, peaks R1 and R3 in Fig. 2B are found to contain two peptides each. Peaks R4, R5, and R6 contained single peptides. If the amounts of two or more peptides are about the same, however, further separation on an17 A. M. Crestfield, S. Moore, and W. H. Stein, J. Biol. Chem. 238, 622 (1963).
[20]
INTERSTITIAL RETINOL-BINDING PROTEIN
211
A 0.8
T1
T2
E P
T3
T4
T5
T6
ii
0.7
Vo 17.2 k
0.6
Vi
o
0.5 c
0.4, 0.3!
.Q
0.2. 0.1 0.0
• .
0
.
.
.
.
50
Fraction
.
.
.
.
.
.
.
.
100 number
u
•
•
150
C
OD280
O D280
•~ - r r
I 25
I MIN
d
cD
I 35
I
I 10
MIN
I 15
FIG. 2. (A) Size fractionation of tryptic peptides of bovine IRBP. Tryptic peptides generated from reduced carboxymethylated bovine IRBP were loaded on a Sephadex G-50 column (1 × 100 cm) and eluted with 0.1 N NH4HCO3. Fractions of I ml were collected. Vo, void volume; V~, included volume. (B) Reversed-phase chromatography ofT3 on a Pep RPC HR 5/5 column. Eluent A, 0.1% trifluoroacetic acid in water; eluent B, 0.1% trifluoroacetic acid in acetonitrile. Flow rate: 0.7 ml/min. Linear gradient 0-50% (B in A) in 30 min. (C) Ion-exchange chromatography of R2 on a Mono Q column. Eluent A, 20 mM Tris-HCl, pH 7.8; eluent B, 1 M NaC! in eluent A. Flow rate: 1 ml/min. Concave gradient, 0-50% (B in A) in 15 min, exponent m = 3.
212
RECEPTORS, TRANSPORT, AND BINDING PROTEINS
[20]
TABLE I AMINO ACID SEQUENCES OF 10 BOVINE IRBP TRYPTIC PEPTIDES PURIFIED FROM FRACTION T3
Peptide Rl.l. R1.2b,, '
R2M1 R2M2 R2M3 R3.1 b R3.2b. C R4 b R5 R6 b
Sequence" IYNRPXXSVSELWTLSQLEGER LXXTSALVLXL SVGAADGSSWEGVGVVPDVAVPAEAALTR ALVIGEVTSGGCQPPQTYHVDDTDLYLTIPTAR SLGPLGEGSXTWEGSGVLPCVGTPAEQALEK SQEILSISDPQTLAHVLTAGVX XASTGEXXXLAGVEPDXXVPM XNIGGPTSSiSALCSYFFXEGPPI EYYTLVDRVPALLSHLAAMDLSSVVSEDDLVTK FDSFADASVLEVLGPYILHQVWEP
a X denotes residues not identified during peptide sequencing or mistmatched residues between the peptide sequence and the deduced amino acid sequence for bovine IRBP. b These sequences were not completed to the terminal R or K. ¢ The sequence is not underlined in Fig. 1.
other column is necessary. We have found that Mono Q columns are a good choice for this purpose. The Mono Q column is packed with a hydrophilic polymer containing quaternary amine groups that remain equally charged over a wide pH range of 2-12. When the major peak (R2) from the reversed-phase column is chromatographed on this anion-exchange column, three peptides are easily resolved, namely, R2M1, R2M2, and R2M3. The sequences of 10 tryptic peptides obtained from T3 are shown in Table I. The same strategy may be applied to the other T fractions. The aminoterminal sequence of the protein can be obtained directly from purified bovine IRBP 18,19 (see underlined amino acid residues 1-32 in Fig. 1). During gel-filtration chromatography, T1 elutes in the void volume. This corresponds to a molecular weight of 30,000 or larger, which cannot be analyzed on the Pep RPC column, which has an exclusion limit of 6,000. Molecular Biology In our original work, s a bovine retina eDNA library in the expression vector hgtl 1 provided by Drs. Khorana and Oprian (Massachusetts Insti~8S.-L. Fong, R. G. Cook, R. A. Alvarez, G. I. Liou, and C. D. B. Bridges, F E B S L e t t . 205, 309 (1986). 19 T. M. Redmond, B. Wiggert, F. A. Robey, and G. J. Chader, B i o c h e m . J. 240, 19 (1986).
[ 2 1]
INTERPHOTORECEPTOR R E T I N O I D - B I N D I N G PROTEIN
213
tute of Technology, Cambridge, MA) was screened with polyclonal rabbit anti-bovine IRBP immunoglobulin G (IgG) and ~25I-labeled protein or horseradish peroxidase-labeled goat anti-rabbit IgG (Bio-Rad, Richmond, CA). The procedure used was described by Young and Davis 2°and Huynh et al. 21 After the k phage was absorbed to the YI090 host cell, 105 plaqueforming units (pfu) was plated on a 150-mm plate. IPTG-containing nitrocellulose filters were overlaid on the plate and saturated with 20% fetal calf serum and 5% nonfat milk. Following incubation with anti-bovine IRBP IgG, the filter was treated with either ~25I-labeled protein A or horseradish peroxidase-labeled goat anti-rabbit IgG. When the human retina cDNA hgtl0 library (provided by Dr. Jeremy Nathans, Johns Hopkins School of Medicine, Baltimore, MD) was screened, nick-translated, 32p-labeled cDNA fragments (bovine probe B23 and H.4 IRBP) were used as probes. The procedure of Maniatis et al. 22 was followed. Phage DNA from positive, isolated clones was purified by the plate lysate method using LambdaSorb (Promega Biotec, Madison, WI). The insert was removed from the phage by E c o R I , purified by agarose gel electrophoresis, and subcloned into M13mpl8 and M13mpl9 for sequencing by the chain-termination method23using the procedure of Dale et al. 24 20 R. A. Young and R. W. Davis, Science 222, 778 (1983). 21 T. V. Huynh, R. A. Young, and R. W. Davis, in " D N A Cloning, A Practical Approach" (D. M. Glover, ed.), Vol. 1. IRL Press, Oxford and Washington, D.C., 1985. 22 T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982. 23 F. Sanger, S. Nicklen, and A. R. Coulson, Proc. Natl. Acad. Sci. U.S.A. 74, 5463 (1979). 24 R. M. K. Dale, B. A. McClure, and J. P. Houchins, Plasmid 13, 31 (1985).
[21] P u r i f i c a t i o n a n d A s s a y of I n t e r p h o t o r e c e p t o r Retinoid-Binding Protein from the Eye B y A L I C E J. A D L E R , GERALD J. CHADER, and BARBARA WIGGERT
Introduction Interphotoreceptor retinoid-binding protein (IRBP) is an extracellular carrier of vitamin A and other lipids. 1-3 It occurs primarily in photosensii A. J. Adler and K. J. Martin, Biochem. Biophys. Res. Commun. 108, 1601 (1982). 2 G. I. Liou, C. D. B. Bridges, S.-L. Fong, R. A. Alvarez, and F. Gonzalez-Fernandez, Vision Res. 22, 1457 (1982). 3 y. L. Lai, B. Wiggert, Y. P. Liu, and G. J. Chader, Nature (London) 298, 848 (1982).
METHODS IN ENZYMOLOGY, VOL. 189
Copyright © 1990by Academic Press, Inc. All rights of reproduction in any form reserved.
