Comp. Biochem. Physiol. Vol. 90B, No. 1, pp. 243-247, 1988 0305-0491/88 $3.00 + 0.00 Printed in Great Britain © 1988 Pergamon Press plc

RIBOFLAVIN-BINDING PROTEIN FROM REPTILES: A COMPARISON WITH AVIAN RIBOFLAVIN-BINDING

PROTEINS

VIRGINIA A. M. ABRAMS, TIMOTHY J. MCGAHAN, JEFFREY S. ROHRER, ANDREW S. BERO and HAROLD B. WHITE, III*

Department of Chemistry, University of Delaware, Newark, DE 19716, USA

(Received 24 June 1987)

Abstract--l. Riboflavin-binding protein (RBP) has been isolated for the first time from reptilian sources. 2. RBP from eggs of Python molurus (Indian python) and Chrysemys picta (painted turtle) has been

isolated and compared to RBP from Gallus gallus domesticus (chicken), a well-characterized protein, and a newly isolated RBP from Cairina moschata (Muscovy duck).

3. Each of the proteins is phosphorylated and glycosylated. 4. The ratio of riboflavin binding to protein is 1 : 1 and the K D for each protein is between 1-3 riM. 5. The mol. wts, different for each species, range from 30,000-40,000, with the reptilian proteins being

approx. 10,000 larger than the avian proteins.

INTRODUCTION

An egg surrounded by a shell is a closed environment for the development of an embryo. All the nutrients including vitamins that are required for the growth of the embryo must be put in the egg prior to laying. The deposition of many vitamins including riboflavin in the oocyte is mediated by specific vitamin binding proteins (White, 1987).

Riboflavin-binding protein was first isolated from the white of chicken eggs (Rhodes et al., 1959) and shown to be a glycophosphoprotein that binds 1 mol of riboflavin per mol of protein with a K o of approx. 1 nM (Becvar and Palmer, 1982). This protein is made in the oviduct and is deposited as part of the albumen as the egg descends the oviduct (Mandeles and Ducay, 1962). In chicken egg white, less than 50% of the riboflavin binding sites are occupied by riboflavin (Lotter et al., 1982; White et al., 1986). Riboflavin-binding protein is also found in egg yolk. Yolk RBP is synthesized in the liver in response to estrogen, secreted into the bloodstream where it picks up riboflavin and transported to the developing oocyte where it is deposited in the yolk as a protein-vitamin complex (Blum, 1967). Both yolk and albumen RBP are coded by the same gene, although yolk RBP lacks 11-13 amino acids at the C-terminus. Post-translational cleavage of 11-13 amino acids at the C-terminus from the serum RBP occurs upon entry to the oocyte (Norioka et al., 1985). The carbohydrate moieties of the two proteins found in the yolk and the albumen are different (Miller et al., 1982a; Hamazume et al., 1984).

The role of RBP in riboflavin transport is quite clear. In eggs laid by hens genetically unable to produce riboflavin-binding protein, the embryos die prior to hatching, unless riboflavin is injected into the eggs (Maw, 1954; Winter et al., 1967). Embryonic death occurs around the 13th day of incubation,

*To whom correspondence should be addressed.

which is about the time that flavokinase levels rise (Zak and McCormick, 1982). Flavokinase is needed to convert riboflavin to the coenzymes FMN and FAD.

The structural features involved in RBP deposition into the oocyte have been examined in the chicken. If phosphoryl groups are removed, there is a 90% decrease in the deposition of the protein into the oocyte (Miller et al., 1982a). Carbohydrate modifi- cation, such as removal of sialic acid residues, affects uptake into the oocyte, but interpretation is difficult (Miller et al., 1981b). It has also been shown that the protein does not require riboflavin bound to it for transport into the oocyte (White et al., 1986).

It is anticipated that important structural features of RBP necessary for function will be retained in evolution of the protein. We have purified riboflavin- binding protein from the egg yolks of two reptilian species, Indian python and painted turtle. Structural features of these RBPs are compared to those of two avian species, chicken and Muscovy duck. The Muscovy duck yolk RBP is described for the first time.

MATERIALS AND METHODS

Materials

Muscovy duck eggs, Cairina moschata var. domestica, were obtained from Dr E. G. Buss, Pennsylvania State University. Turtle oocytes, Chrysemys picta, were a gift from Dr Gregory Stephens, School of Life and Health Sciences, University of Delaware. Python molurus eggs were obtained from Dr Dale Marcellini, Curator of Herpetology, National Zoological Park, Washington, D.C.

Protein purifications

Initial purifications of each of the egg samples were carried out using the method described by Miller and White (1986) for chicken RBP. These procedures did not yield pure protein from Muscovy duck, painted turtle, or Indian python egg yolk. Chromatography on an N-3-carboxy- methylriboflavin-substituted agarose affinity column

243

244 VIRGINIA A. M. ABRAMS et al.

(Merrill and McCormick, 1978) succeeded in separating the riboflavin-binding protein from the impurities.

Special care was taken to preclude copper contamination of the proteins by using deionized distilled water, boiling dialysis tubing in 30 mM EDTA and using 3 mM EDTA in all buffer preparations. Final dialyses were done using deionized, distilled water containing Chelex 100 (Bio-Rad.). Values as low as 0.5 Cu atoms/mol of RBP slowly denatured the apoprotein even at freezer temperatures. The protein would turn pale blue or green, become glossy in appearance and lose riboflavin binding capacity. Removal of copper to levels below detection using Chelex 100 did not completely restore activity to the protein.

Analytical methods

Protein concentrations were calculated using the method of Lowry et al. (1951). Absorbance at 280 nm was routinely used to determine RBP concentration using a Lambda 4B Perkin Elmer UV/Vis Spectrophotometer. A Turner Fluorometer was used to measure quenching of riboflavin fluorescence by the apo protein which, in turn, was used to determine apo-RBP concentrations, and to calculate K D values (Becvar, 1973). Phosphoryl group analysis was done by the method of Ames (1966) and sialic acid determination using the method of Warren (1959) and that of Skoza and Mohos (1975). Neutral sugars were determined using the orcinol-H2SO 4 method of Francois et al. (1962). Carbo- hydrate analysis by gas chromatography (Perkin Elmer 3920B, Supelco SP 2340 column packing) was carried out using alditol acetate derivatives (Porter, 1975). Protein content for the alditol acetate assay procedure was mea- sured with Pierce BCA Protein Assay (Pierce Application Sheet No. 23225). Copper analyses using neocuproine were done using the method of Diehl and Smith (1958). Disulfide groups in the proteins were determined in the manner of Cavallini et al. (1966). Free sulfhydryl groups were assayed using the method of Habeeb (1972). Tryptophan content of the proteins was determined by the method of Spies and Chambers (1948). Amino acid analysis was done with a Waters Company Pico-tag Amino Acid Analyzer.

Gel electrophoresis

SDS polyacrylamide gel electrophoresis (SDS PAGE, 10% gels, room temperature) were run in the manner of Weber and Osborn (1969). Commercial standards (Bio-Rad SDS PAGE 10,000-100,000 mw standards) were used for mol. wt estimation. Polyacrylamide gel electrophoresis (PAGE) analysis (7% gels, room temperature) was done as described by Weber and Osborn (1964).

Electrophoresis studies o f turtle yolk RBP

Turtle yolk RBP showed two bands on analysis with PAGE, although only one band on SDS-PAGE. Samples were treated to remove possible sources of charge hetero- geneity and examined by PAGE. Dephosphorylation of the protein (I mg) was done with potato acid phosphatase (Boehringer-Mannheim, 60 U/ml) as described by Miller et al. (1982a). Sialic acid residues were cleaved by heating the protein (1 mg) in 0.1 N H2SO 4 (Miller et al., 1981b). One sample was treated first with potato acid phosphatase and then dilute sulfuric acid to remove both phosphate and sialic acid residues. Another sample of turtle yolk apo-RBP was treated prior to electrophoresis with equimolar [2-14C] riboflavin (Amersham, 1 #C/ml). The gel was run, but not stained, because riboflavin will be released at the low pH of the methanol-acetic acid destaining solution. A stained gel of turtle yolk RBP was used as a guide and the gel was cut into 3 mm pieces for counting on a Beckman Liquid Scintil- lation Counter. Pieces were taken from the two protein bands and the areas before, between, and after the bands. Gel pieces were placed in counting vials, treated with 1 ml of a 9:1 solution of Protosol (New England Nuclear) and water, and heated for 18 hr at 37°C. After l0 ml of scintil-

lation fluid was added, samples were counted in the Liquid Scintillation Counter.

RESULTS AND DISCUSSION

Yolk riboflavin-binding proteins have been iso- lated from reptilian sources, Indian python eggs, and painted turtle oocytes, and from Muscovy duck eggs for the first time. Purity of the proteins was estab- lished by showing that each protein showed one band on SDS-PAGE gels. With P A G E gels, the duck and python RBP proteins gave one band, but the turtle protein resolved into two bands (see below). Solu- tions of each of the apo proteins quenched the fluorescence of free riboflavin, demonstra t ing binding of the riboflavin, which is known to procede with quenching of the riboflavin fluorescence.

The mol. wt of chicken yolk RBP is known from its amino acid sequence and carbohydrate com- position to be 30,000 (Norioka et al., 1985). Apparen t mol. wts of the newly isolated proteins and a chicken RBP standard, estimated by SDS-gel electrophoresis, showed that the smallest o f the riboflavin-binding proteins was chicken (40,000) followed by duck (42,000), python (49,500) and turtle (50,000). Glyco- proteins are known to give aberrant mol. wt es t imates with SDS gels (Leach et al., 1980) as are phospho- proteins (Simmons, 1984). Using the known mol. wt of chicken yolk RBP and assuming similar electro- phoretic behavior, the mol. wts of the three new proteins were estimated to be 32,000 for Cairina moschata , 39,500 for P y t h o n molurus and 40,000 for Chrysemys p ic ta (Table 1). Values of protein concen- tration of the newly isolated proteins were calculated from the absorbance at 280 nm using a molar extinc-

Table 1. Amino acid composition (mol %) of chicken, duck, turtle and python yolk RBP

Amino Muscovy Painted Indian acid Chicken* duck'? turtlet pythont

asp 10.7 10.8 14.1 12.0 glu 15.8 16.4 13.7 15.4 ser 16.4 17.4 18.3 16.3 his 4.4 3.5 4.7 2.5 gly 3.3 5.1 5.0 5.0 thr 3.8 4.1 4.2 5.5 ala 6.6 5.5 5.5 6.6 arg 2.7 2.3 2.9 4.6 tyr 4.9 5.9 4.8 3.6 val 2.7 3.3 3.9 2.4 met 3.8 3.9 4.1 4.2 ile 3.8 2.6 2.8 4.3 phe 2.7 3.2 2.5 2.3 leu 6.0 6.7 5.4 6.1 lys 7.8 7.2 5.3 5.7 pro 4.4 3.9 4.1 3.5 1/2 cyst: 18.0 12.2 10.1 10.8 trp$ 6.0 7.2 6.5 4.8

*Values were calculated from amino acid sequence reported by Norioka et al. (1985).

tA Waters Pico-Tag Amino Acid Analyzer was used. Samples were hydrolyzed for 22 hr in HC1 vapor and then derivatized with phenyl isothiocyanate in the instrument. The derivatives were analyzed by reverse phase HPLC. Neither tryptophan, cysteine nor cystine can be quantitated by this method. Amino acid amounts are reported in mol % of amino acids measured.

SValaes for tryptophan and cysteine were determined separately (see Analytical Methods Section) and are expressed as mol/mol RBP rather than mol %. Half-eystine experimental values were all corrected to the known value of chicken ( x 1.23).

Reptiles riboflavin-binding proteins 245

I0

9

8

7

6 _~:~

~ 4

3 D

I I L I I I 20 40 60 80 IO3 120

I [108/M ] [Rfl

140

Fig. 1. Scatchard plot of the titration of yolk apo-RBP for duck (I--I), turtle (A) and python (A) with riboflavin.

tion coefficient of 49,000/M, the value determined for chicken RBP, Becvar (1973). These values compared well with protein concentrations determined by the Lowry method (Lowry et al., 1951).

The dissociation constant, Ko, of riboflavin for each of the newly isolated proteins is in the same range as that of chicken RBP. The KD values were measured by following the fluorescence of a dilute solution of apoprotein as known aliquots of riboflavin were added. In Fig. 1, the fluorescence data are plotted in a linear Scatchard relationship, where K o can be obtained from the slope.

1 KD 1 1 -- X

(RBP 'Rf ) (RBP)0 ~ + (RBP)0

(RBP. Rf) is the total amount of the complex in the system, (RBP)o is the initial concentration of apo- RBP, and (Rf) is the concentration of free riboflavin. The slope is KD/(RBP)o and the intercept is I/(RBP)o. Values for the dissociation constant, determined from data in Fig. 1, were 1.72 + 0.7 nM for Muscovy duck, 3.2 + 0.2 nM for Indian python, and 3.2 + 0.2 nM for painted turtle.

Measuring the amount of riboflavin quenched by a known amount of apo-RBP (calculated from absorbance at 280 nm) showed the activity of the proteins was a little more than 50% of that expected for a one to one binding ratio of protein to riboflavin. The low activity may be due to the harsh conditions (pH 3.5) that were used to remove the bound protein from the affinity column. The age of the proteins at the time of comparative analysis (1-4 y) could also be a factor in decreased activity. We have observed a considerable decrease in the activity of apo-chicken yolk RBP with age.

Amino acid analyses are reported as mol % in Table 1. Glutamic acid, aspartic acid, serine and lysine are the most plentiful residues in these proteins, which is also the case in the well documented chicken RBP. Chicken yolk RBP has 18 cysteine residues, which account for the 9 disulfide bonds reported by Hamazume et al. (1984). Muscovy duck and Indian python RBP appear to have 12.2 and 10.8 sulfhydryl groups, respectively (Table 1). Muniyappa and Adiga (1980) report 12.6-SH bonds (or 6 disulfide bonds) in Anas platyrhynchos egg white RBP. No free cysteines were detected in any of the proteins. Tryptophan was present in each of the new proteins.

Phosphoryl group analysis (Table 2) showed that each of the proteins, as in the case of chicken RBP, is phosphorylated. Neutral sugar analysis confirmed that the proteins are all glycosylated (Table 2). Each of the new proteins contained sialic acid residues (Table 2). Sialic acid residue figures may be arti- factually low because the sialic acid glycosidic linkage is cleaved quite readily in acidic solutions (Sharon, 1975) and some sialic acid may be lost during acidic dialyses during protein purification.

Neutral sugar and N-acetyl glucosamine content was determined for the Muscovy duck and python yolk RBP samples by gas chromatography of alditoi acetate derivatives. As can be seen in Table 3, Muscovy duck and chicken are quite similar in carbohydrate content. On the other hand, python yolk RBP appears to have a considerably larger amount of carbohydrate. Turtle yolk RBP was not analyzed due to uncertainties about heterogeneity as seen on non-denaturing gel electrophoresis. Chicken yolk RBP has two complex, N-linked oligosaccha- rides (Miller et al., 1981a). Because the sugar com- position of the Muscovy duck RBP oligosaccharide

Table 2. Molecular weight, phosphate and sugar compositions of yolk RBP from chicken, Muscovy duck, painted turtle, and Indian python

Values/mol of RBP

Species Mol. wt - PO 3 = Hexose Sialic acid

Gallus gallus domesticus 30,000 8.9 l l.0 3.5 Cairina moschata domestica 32,000 7.5 10.4 2.5 Chrysemys picta picta 40,000 5.6 20.6 5.1 Python molurus 39,500 8.6 13.5 7.4

Table 3. Carbohydrate analyses of yolk RBP from chicken, duck and python by gas chromatography of alditol acetate derivatives

Species Mannose Galactose N-Acetyl glucosamine

Gallus gallus domesticus* 5.9 + 0.l 4.9 + 0.1 12.0 _+ 2.3 Cairina moschata domestica 5.8 _+ 0. I 3.6 + 0.1 10.2 _4- 1.1 Python molurus 8.5 _+ 0.l 7.9 + 0.4 22.7 + 1.6

*Values for chicken are taken from Miller et al. (1982b).

246 VIRGINIA A. M. ABRAMS et al.

is so similar to that of chicken RBP, it is probable that it also has two attachment sites with oli- gosaccharides of similar structure. It has been re- ported that egg white RBP from another duck, Anas platyrhynchos, is not a glycoprotein (Muniyappa and Adiga, 1980). As shown here, Muscovy duck clearly is a glycosylated protein, and in contrast to the earlier report, we have found that egg white RBP from Anas platyrhynchos (White Pekin) also contains carbo- hydrate (Lisa Meyer, unpublished). The amount of carbohydrate associated with the python protein is much greater, and it is possible that there are three or more attachment sites.

Concern about the two bands shown on non- denaturing gels by the turtle protein caused us to look for the source of the apparent heterogeneity in the protein. Because the turtle protein was purified by riboflavin affinity chromatography, it seemed likely that two forms of RBP were present. Riboflavin binding by the two protein bands was tested by gel electrophoresis with an equimolar amount of [2J4C] riboflavin added to the turtle apo-RBP sample. Both of the protein bands bound riboflavin with the less anionic form constituting three quarters of the mix- ture (Fig. 2). Because both proteins bound riboflavin and their mol. wts are 40,000 -+ 4000, it is likely that the two proteins are forms of RBP that differ in charge. Experiments were performed to determine if the difference in electrophoretic mobility was due to differences in the numbers of phosphoryl and/or sialic acid residues. Some broadening and slowing of the faster moving (more negative) band was observed both when the protein was treated to remove phos- phoryl groups and when the protein was treated to cleave sialic acid residues. Maximum slowing and broadening occurred when both treatments were used on the protein, but the bands did not coalesce, suggesting that the two RBPs may be isoprotein products of homologous genes, although other possi- bilities exist. One possibility is that the two forms of the protein differ by a post-translational modification like the cleavage of the chicken serum RBP to form the chicken yolk RBP, where the last 11-13 amino acids are cleaved from the carboxyl end of the pro- tein (Norioka et al., 1985). This 11-13 amino acid polypeptide contains 5 glutamic acid residues which

mm - - 0

Stacking / ~ ] - - 1 3 Gel

- - s 2 - - 5 6

~ - - 7 3 - - 7 8

Tracking ~ --104 Dye ~ mm

Counts from Gel Tube Background = 34.3 cpm

801

L 3 1 1

Fig. 2. Polyacrylamide gel electrophoresis of turtle yolk RBP bound to 2 -14 C riboflavin, showing position of the two bands and the counts per minute from the radioactive

riboflavin associated with each.

account for a difference of 5 negative charges in the yolk and serum proteins. Another possibility is that the forms are products of allelic genes for RBP, that differ in electrophoretic properties. The turtle oocytes were collected from a number of individuals. The two forms of the turtle protein have not been isolated in quantities large enough to distinguish among these alternatives.

In so far as is known, all vertebrate embryos require riboflavin. One would expect that once a mechanism developed to supply riboflavin to the embryo, it would be retained in subsequent species during evolution. We have shown here that a turtle and a snake, represented by Chrysemys picta and Python molurus, respectively, do have riboflavin- binding proteins with characteristics quite like those of birds, such as Gallus gallus domesticus and Cairina moschata. All are phosphoglycoproteins with mol. wts of 30,000-40,000 that bind riboflavin with a K o of 1-3 nM. Because the phosphate and carbohydrate features are preserved in the different species, they may be necessary for the function of the protein and transport of the vitamin from the blood into the developing oocyte.

Acknowledgements--We thank Drs Dale Marcellini, Gre- gory Stevens, and Edward Buss for oocytes and eggs used for the isolation of riboflavin-binding proteins. We also thank Dr Lloyd Abrams for his assistance in KD calculations and in the manuscript preparation, as well as Larry Bush for running the tryptophan analyses. This research was supported in part by USPHS grant AM 27873 and the University of Delaware Undergraduate Honors Program.

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