Hydatigera taeniaeformis: Strobilocerci hooks

EXPERIMENTAL

26, 128-133

PARASITOLOGY

Hydatigera III.

Molecular

( 1969)

faeniaeformis:

Weight

and N-terminal James

Department Uniz;ersity

Strobilocerci Amino

Hooks

Acid

Determinations

A. Dvorak1

of Animal Biology,2 School of Veterinary Medicine, of Pennsylvania, Philadelphia, Pennsylvania 19104

(Submitted

for publication,

24 January

1969)

DVORAK, JAMES A. 1969. Hydutigera taeniaeformis: Strobilocerci hooks. III. Molecular weight and N-terminal amino acid determinations. Experimental Purasitology 26, 128-133. Rostellar hooks of the strobilocerci of Hydatigera taeniueformis, Batsch, 1786, solubilized by cystine disulfide bond reduction yielded a heterogeneous mixture of proteins with a maximum molecular weight of 8-10,000. N-terminal amino acid analysis of the hook protein molecules revealed the presence of a single N-terminal amino acid, cystine. It is postulated that the rostellar hooks of H. taeniaeformis strobilocerci are polymer-like structures with cystine disulfide linkage of the heterogeneous protein subunits.

INDEX DESCRIPTORS: Hydutigeru taeniaeformis, tapeworms; cestodes; amino acids; physiology.

The major organic component of cestode hooks is a cystine-rich, keratin-like protein (Crusz, 1947,1948; Gallagher, 1964; Dvorak, 1969a). The rostellar hooks of Hydatigera taeniaeformis, Batsch, 1786, strobilocerci also appear to contain a large quantity of labile nonprotein sulfur and a small quantity of lipid (Dvorak, 1969a,b). Solubility studies indicate that the lipid component may act as a barrier which hinders solubilization of the hooks by disulfide bond reduction. After lipid extraction, however, the hooks can be solubilized by selective cleavage of cystine disulfide bonds with 2-mercaptoethanol. Although the hook protein appears to be electrophoretically nonhomogeneous, this in1 This investigation was supported in part by the Molecular Biology Training Grant, USPHS 5 TO l-GM-00694-07, Graduate Group on Molecular Biology, Graduate School of Arts and Science, University of Pennsylvania. 2 Present address: National Institutes of Health, National Institute of Allergy and Infectious Diseases, Laboratory of Parasitic Diseases, Bethesda, Maryland 20014.

strobilocercus,

hooks; protein;

cystine;

homogeneity probably is a reflection of the complex nature of the hook and not an artifact caused by the solvent. Attempts were made, therefore, to determine the molecular weight of the hook protein molecules. The high concentration of cystine in the hooks raised the question as to whether some disulfide bonds might not be present as polymerlike intermolecular linkages of the hook protein molecules. N-terminal amino acid analysis of the hook protein was undertaken in an effort to resolve this question. All of the determinations described in this paper were performed on a sample of clean hooks (9.38 * 0.015 mg) which had been pretreated with hot pyridine and solubilized with 1 M 2-mercaptoethanol3 M urea as described previously (Dvorak, 196913). MATERIALS

Molecular

AND METHODS

Weight Determination

Ultracentrifugation and light were used to determine the 128

scattering molecular

STBOBILOCERCI

weight of the protein molecules. Sedimentation velocity ultracentrifugation followed the method described by Schachman (1957); the sample was contained in a standard sector cell with a Kel-F centerpiece and was centrifuged at 56,100 rpm at 20.8”C for 75 minutes in a Beckman Model E analytical ultracentrifuge. Schlieren optics were used to view the cell during ultracentrifugation. An aliquot of the hook protein solution was serially diluted with solvent and measured with the Brice-Phoenix universal light-scattering photometer described by Brice et al. ( 1950). Light scattered by the hook protein solutions and the solvent was measured with the photomultiplier at 90” from the incident beam; transmitted light was measured with the photomultiplier at 0”. Five readings were made at the 90” and 0” positions for each solution. N-terminal

Amino Acid Analysis

An effort was made to determine the Nterminal amino acid groups present in the hook protein solution using the ‘dansyl’ method of Gray and Hartley ( 1963). Dansyl reagent (1-dimethylamino-naphthalene-5sulfonyl chloride (DNS) in acetone, 1 mg/ ml) was added to an aliquot of the original hook protein solution. The mixture was shaken for 1 hour at room temperature after which 6% trichloroacetic acid was added to precipitate the protein. A white flocculant precipitate appeared which was washed three times with methanol: diethyl ether (3: 1) followed by two washes with diethyl ether. After the ether had been removed, 6 N HCl was added to the precipitate, the tube was sealed, and the protein hydrolyzed at 100°C for 12 hours. After hydrolysis, the hydrolyzate was dried in ~XLCUO over KOH pellets. The residue was dissolved in 0.01 N HCl in preparation for thin-layer chromatography on Silica G plates using solvent systems described by Zdenek and Rosmus ( 1965). DNS derivatives were detected by their yellow fluorescence in UV light.

HOOKS.

129

III

RESULTS

Molecular

Weight

Determination

The hook protein did not sediment after 75 minutes centrifugation at a speed of 56,100 rpm; however, a marked thickening at the meniscus of the solution in the cell appeared (Fig. 1). It is possible that this thickening of the meniscus represented protein which might have sedimented at a higher centrifugal force. The molecular weight of the hook protein could be determined from light-scatter data (Tanford, 1961). A reasonably straight line resulted from a plot of He/t 0s. c (Fig. 2, Table I). Extrapolation to zero protein concentration yielded a maximum molecular weight of 8000 assuming the hooks are pure protein. A second calculation of molecular weight based on the percentage of nitrogen recovered from amino acid analyses (82% nitrogen recovery (Dvorak, 1969a) ) resulted in a maximum molecular weight of 10,000. The second virial coefficient, B, which is the slope of the He/t OS. c plot can be used to estimate the excluded volume of the solute particles (Tanford, 1961). If it is assumed that the solute particle is a rigid rod with a diameter of 12 A (based on high resolution electron microscopy observations (Dvorak, unpublished data) ) the calculated maximum length of the molecule is 114 A (assuming the hooks are pure protein) to 144 A ( assuming the hooks are 82% protein). N-terminal

Amino Acid Analysis

In a two-dimensional chromatographic separation using benezene:pyridine:acetic acid (40: 10: 1) as a solvent in the first direction and chloroform: benzyl alcohol:acetic acid (70:30:3) as a solvent in the second direction, only slight movement of the fluorescent DNS derivative from the origin resulted (Fig. 3A). According to Zdenek and Rosmus (1965) the only common DNS amino acid derivative which remains at the

130

DVORAK

FIG. 1. Ultracentrifuge cell containing solution of hooks solubilized with 2-mercaptoethanol. Photograph made 75 minutes after reaching a speed of

56,100 rpm in a sedimentation velocity run. Top of cell is at left; bottom of cell at right. Note the marked thickening at the meniscus (center).

origin with both of these solvents is DNS cysteic acid; however, the DNS cysteic acid derivative does migrate in other solvent systems. In N-butanol:pyridine:acetic acid: water (15:10:3:12) an RF value of about 0.5 is expected and in chloroform: ethanol: acetic acid (38:4:3) an RF value of about 0.3 is expected. In a second two-dimensional using Nchromatographic separation butanol:pyridine:acetic acid:water as a solvent in the first direction and chloroform: ethanol: acetic acid as a solvent in the second

OO

I 0.2

I 0.4

I 0.6

PROTEIN, em/ml

I 0.6

I 1.0

(~10~~)

FIG. 2. A plot of the He/t values obtained by light-scatter VS. protein concentration assuming the hooks are 100% protein. Extrapolation to zero protein concentration yields a molecular weight of 8000.

TABLE I LighMcattering Data for the Solutions of Solubilized Hooks of Hydatigera taeniaeformis Assuming the Hooks to be Pure Protein Protein concn gmhl (X 10-3) 0.938 0.469 0.235 0.117

HC/T ( x 10-4) 8.95 5.42 3.45 1.95

STROBILOCERCI HOOKS. III

Benzene:Pyridinc:HAc

131

n-Butanol:Pyridinc:HAc:H20

____________--__________

D .

n-Butanol:Pyridinc:HAc:H20

.

.

0

0

n-Butanol:Pyridine:HAc:H20

FIG. 3. Schematic representation of chromatography of the DNS derivatives obtained from the protein of H. taeniaeformis strobilocerci hooks. Open circles indicate origins, dark circles indicate fluorescent spots, broken lines indicate solvent fronts. In D the dark circle to the left of center indicates the DNS derivative obtained from hook protein; the dark circle to the right of center indicates the DNS derivative of cysteic acid.

direction, all of the fluorescent material left the origin and appeared as a single spot in about the position predicted for DNS cysteic acid (Fig. 3C). A third two-dimensional chromatographic separation was tried using N-butanol:pyridine:acetic acid:water as a solvent in the first direction and chloroform: benzyl alcohol:acetic acid as a solvent in the second direction. All of the fluorescent material left the origin and appeared as a single spot with an RF value of about 0.5 from the first solvent and unaffected by the second solvent ( Fig. 3B ) . In order to confirm the identity of the fluorescent spot, a DNS ,cysteic acid derivative was prepared and chromatographed simultaneously with the sample. One-dimensional chromatography was performed with N-butanol:pyridine:acetic acid:water as the solvent. Both the DNS cysteic acid standard

and the sample appeared to have migrated the same distance with respect to the solvent front (Fig. 3D). Thus, a single N-terminal amino acid was detected which, on the basis of its migration in various solvent systems and, compared to a DNS cysteic acid derivative, appeared to be cysteic acid. DISCUSSION

If the hooks of H. taeniaeformis strobilocerci were composed of a homogeneous protein, the minimum molecular weight of the protein could be estimated from amino acid analysis. Based upon methionine, which is present in a concentration of about 0.1 gm/lOO gm of hooks (Dvorak, 1969a), the molecular weight of the protein would be about 150,000. This value is obviously invalid for hooks solubilized by disulfide bond

132

DVORAK

reduction into a heterogeneous mixture of subunits. A protein with a molecular weight of 150,000 would have sedimented in the ultracentrifuge at the speeds used. The maximum molecular weight value of 8000 to 10,000 as determined by light-scattering measurements probably represents the value of the largest molecules present in the pyridine-extracted, 2-mercaptoethanol solution of hooks. The weight and size values (molecular weight of S-10,000, length of 114144 A, width of 12 A) are similar to those obtained for wool keratin which, when solubilized with sodium sulfite, exhibits polydispersity with an average molecular weight of 9000, an average length of 170 A, and a width of 11 A (Matoltsy, 1962). The presence of cystine as an N-terminal group has been reported for oxytocin and vasopressin (duvigneaud et al., 1954) and chymotrypsin (Bettelheim, 1955). Such a group could increase the structural strength of the cestode hook through polymer-like cross-linking of adjacent peptides or protein molecules and provide a means of generating a morphologically complex hook structure from heterogeneous subunits. The breakage of these bonds by disulfide reduction would result in the appearance of a heterogeneous mixture of low molecular weight protein molecules (Turner et d., 1959) as was observed in this study. The presence of cystine disulfide linkages at sites other than N-terminal in the molecules can only be speculated on. However, the breakage of intrachain cystine disulfide bonds, which occur commonly in protein structures, would not result in lowering the molecular weight of the protein molecules. At this point, some consideration can be made of the relationship between the structure of the adult cestode hook and its function. The primary function of the hook is to help hold the cestode in the host, a function which must be performed without evoking a severe response on the part of the host. This necessitates that the hook be struc-

turally strong yet inert in the sense that it does not display its “foreignness” to the host. A hook composed of short peptide subunits bound in a polymer-like configuration by cystine disulfide bonds would provide an excellent structural “backbone.” A marked decrease in the elasticity inherent in such a keratin-like structure could be obtained by the incorporation of additional sulfur Ii&ages producing an effect similar to that observed when excess sulfur is added to raw rubber during the process of vulcanization (i.e., elastic properties disappear and hard rubber or ebonite results). The antigenie potential of the hook could be decreased or extinguished both by masking or neutralizing chemically active sites on the hook protein and also by covering the structure with an antigenically less active lipid. The hook, which is the cestode structure in most intimate contact with host tissues, may utilize all of these mechanisms to perform its function successfully. ACKNOWLEDGMENTS The author expresses appreciation to Dr. I. Gersh and Dr. M. R. Iyengar for permitting full use of their laboratory facilities and for helpful discussions during the course of this work.

REFEHESCES RETTELHFIM, F. R. 1955. Amino terminal group in chymotrypsin. Journal of Biological Chemistry 212, 235-239. BRICE, B. A., HALIVER, M., A\TD SPEISER, R. 1950. Photoelectric light-scattering photometer for determining high molecular weights. iournal of the Optical Society of America 40, 768-778. CHUSZ, H. 1947. The early development of the rostellum of Cysticercus fascioluris Rud., and the chemical nature of its hooks. Journal of Parasitology 33, 87-98. CIWSZ, H. 1948. Further studies on the development of Cysticercus fasciolaris and Cysticercus pisiformis, with special reference to the growth and sclerotization of the rostellar hooks. Journal of HeZminthoZogy 22, 179-199. DVORAK, J. A. 1969a. Hydatigera taeniaeformis: Strobilocerci Hooks. I. Collection and pre-

STROBILOCEHCI paration of hooks; elemental, amino acid and infrared spectrophotometric analyses. Experimentd Parasitology 26, 111-121. DVORAK, J. A. 1969b. Hydatigera taeniaeformis: Strobilocerci Hooks II. Solubility and Structural Homogeneity. Experimental Parasitology 26, 122-127. GALLAGHER, I. H. C. 1964. Chemical composition of hooks isolated from hydatid scolices. Experimental Parasitology 15, 110-117. GRAY, W. R., AND HARTLEY, B. S. 1963. A fluorescent end-group reagent for proteins and peptides. Biochemical Journal 89, 59p. MATOLTSY, A. G. 1962. Structural and chemical properties of keratin-forming tissues. In “Comparative Biochemistry,” Vol. 4, pp. 343-369. Academic Press, New York.

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133

SCHACHMAN, H. K. 1957. Ultracentrifugation, diffusion and viscometry. In “Methods in Enzymology,” Vol. lV, pp. 32-103. Academic Press, New York. TANFORD,~. 1961. “Physical Chemistry of Macromolecules,” Academic Press, New York. TURNER, J. E., KENNEDY, M. B., AND HA~RO~Z, F. 1959. Disulfide bonds in proteins. In “Sulfur in Proteins,” pp. 25-31. Academic Press, New York. VIGNEAUD, V. DU, RESSLER, C., SWAN, J. M., ROBERTS, C. W., AND KATSOYANNIS, P. G. 1954. The synthesis of oxytocin. Journal Of the American Chemical Society 76,3115-3121. ZDENEK, D., AND ROSMUS, J. 1965. Thin layer chromatography of dansyl amino acid deriva20, 514-520. tives. Journal of Chromatography