Identification of a novel parvalbumin in avian thymic tissue

Vol. 177, No. 2, 1991 June 14, 1991

BIOCHEMICAL

IDENTIFICATION

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 881-887

OF A NOVEL PARVALBUMIN

IN AVIAN THYMIC TISSUE

Michael T. Henzl, Rita E. Serda, and Jeanne M. Boschi

Department Received

of Chemistry, New Mexico State University Las Cruces, NM 88003

May 8, 1991

A novel calcium-binding protein has been isolated from chicken thymus tissue. Its molecular weight (=I 1,500) and characteristic interactions with Tb3+ and Eu9+ identify the protein as a member of the parvalbumin family. Electrophoretically distinct from both chicken (muscle) parvalbumin and avian thymic hormone, it represents the third parvalbumin to be identified in avian tissues and the second to be identified in the avian thymus gland. 0 1991 Academic Press, Inc. The parvalbumins Possessing

are vertebrate-specific

two high-affinity

parvalbumins

Ca2+-binding

have traditionally

at highest levels in skeletal muscular

and neuronal

kidney, adipose conjectural,

(3,4).

to the prevention

that a thymic protein

evidence

suggested

another

basis of molecular previously parvalbumin

Ragland

parvalbumin

for stimulating

in the

protein,

protein

leg muscle. to three.

distinct

(11).

capability

from

with the The suggests

the

of this class of proteins.

expressed

luminescence

isolated

has been confirmed

scheme for ATH from chicken

it is electrophoretically

is increased

was, in

(ATH), has been sequenced

at substantially

in purifying the latter protein to apparent

weight and lanthanide

repertoire

is still

T-cell maturation

with immunoregulatory

roles for members

of a purification

from chicken

in

and his colleagues

for the muscle-associated

small calcium-binding

However, isolated

differentiation,

earlier (lo), and the nonidentity

physiological

We recently succeeded

parvalbumin.

is also produced

that ATH was distinct from the parvalbumin

of a thymus-specific of additional

During the development

ATH.

to participate

the entire coding region has been cloned (9).

recent report of a partial cDNA sequence

we detected

They are expressed

and are believed

(7). This protein, called avian thymic hormone

leg muscle a decade

identification

Ca2+ buffers.

of Ca2+ toxicity (6).

having the capacity

spanning

(1,2).

referred to as the CD and EF sites,

In the rat, parvalbumin

avian T-lymphocyte

(8), and a cDNA fragment

existence

superfamily

tissue, and testis (5). The function of the protein in these tissues

fact, a parvalbumin

chicken

sites, generally

muscle fibers and certain neurons

relaxation

While investigating

Circumstantial

of the calmodulin

been viewed as cytoplasmic

but may be related

discovered

members

properties,

thymus tissue (12), lower

levels than

homogeneity.

it appears

On the

to be a

from both ATH and the parvalbumin

Thus, with this finding,

membership

in the avian

Vol.

BIOCHEMICAL

177, No. 2, 1991

AND BIOPHYSICAL

MATERIALS

RESEARCH COMMUNICATIONS

AND METHODS

Chicken leg muscle and thymus glands were obtained from Pel-Freez, Inc., and were stored at -70” C prior to use. 45Ca2+ was purchased from New England Nuclear. TbCl3 and EuCl3 were purchased from Aldrich Chemical Co. DEAE-Sepharose CL-GB, Sephadex G-75, and Sephadex G-100 -- products of Pharmacia, Inc. -- were obtained from Sigma Chemical Co. A Mono Q FPLC column (HW55) was purchased directly from Pharmacia. All other reagents and chemicals were obtained from Sigma. Discontinuous SDS-PAGE was performed using the buffer system developed by Laemmli (13), and proteins were visualized with Coomassie Brilliant Blue R250. Electrophoretic transfer of proteins from SDS polyacrylamide gels to nitrocellulose membranes was carried out for 50 minutes in a Bio-Rad Trans-Blot@ cell at an electric field of 8 V/cm in 0.025 M Tris, 0.20 M glycine (pH 8.3) containing 20% (v/v) methanol. Protein concentrations were determined with the Pierce protein assay reagent, employing the bovine serum albumin supplied with the reagent as a standard. Tb3+ luminescence was measured on an Aminco spectrofluorimeter. The bound Tb3+ ions were excited indirectly (hex=260 nm), relying on energy transfer from adjacent phenylalanyl residues. Nominal excitation- and emission bandwidths of 5.5 nm and 11 nm, respectively, were employed. The sample was housed in a 3 mm (I.D.) cuvet, to avoid complications arising was measured with the laser from the inner filter effect. Eu 3+ luminescence spectrofluorimeter described elsewhere (14,15). The Eu3+ 7Fo+5Do transition near 580 nm was excited directly with a nitrogen-pumped tunable dye laser, employing Rhodamine 6G. Luminescence, resulting from the 7Fo+5D2 transition, was monitored at 615 nm. Avian thymic hormone was isolated by the method described previously (12). The parvalbumin from chicken leg muscle was purified by a minor modification of the procedure outlined by Strehler et al. (10). Isolation of the novel calcium-binding protein from thymus tissue involved heat-treatment of the crude extract at 80” C, ion-exchange on DEAE-agarose at pH 7.4, gel-filtration on Sephadex G-75, and ion-exchange on Mono Q at pH 5.7 and at pH 7.4. The initial purification was monitored by 45Ca2+ autoradiography on nitrocellulose membranes following SDS-PAGE (16). Subsequent isolations were then monitored electrophoretically, employing the previously purified material as a standard. The material employed in the lanthanide luminescence experiments described below contained 2.0 equivalents of Ca2+, as determined by flame atomic absorption spectrophotometry. RESULTS A novel calcium-binding homogeneity

by a modification

Like ATH, it is remarkably Although

protein

heat-stable,

employed

surviving

or Mono Q columns.

thymus was purified

slightly

(12).

at 80” C for five minutes.

G-75, they can be separated

requiring

to apparent

to isolate avian thymic protein

heat-treatment

from Sephadex

with the novel protein

from DEAE-agarose

from chicken

of the procedure

the two proteins co-elute

chromatography,

(CaBP)

higher

by ion-exchange

ionic strength

The yield of the purified

for elution

novel CaBP -- typically

l-2

mg from 200 g of crude thymus tissue -- is some five to ten times lower than that of ATH. The ultraviolet indicates

an absence

reminiscent Although hormone

spectrum

of tryptophan

of those previously

from chicken SDS-PAGE.

absorption

of the purified

protein,

displayed

and a high PHE: TYR ratio. The spectrum

reported

in Figure

1,

is, in fact, highly

for ATH (12) and the muscle-associated

parvalbumin

(10). very similar

spectroscopically,

Figure 2A compares (lane 1) displays

the three proteins

their electrophoretic

a mobility

consistent

mobilities

can be readily distinguished in a 14% gel.

with its sequence-derived

882

by

Avian thymic

molecular

weight

of

Vol. 177, No. 2, 1991

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

B

A 1

01

260

300 WAVELENGTH

Figure

1.

Figure 2. from chicken.

2

3

4

2

1

3

340 (NM)

Ultraviolet

absorption

spectrum

Relative electrophoretic A) SDS-PAGE. 1.0

of

mobilities

the of

novel

thymic

three

CaBP.

Can+-binding

proteins

pg samples of ATH (lane 7) the novel thymic CaBP (lane 2), and chicken (muscle) parvalbumin (lane 3) were subjected to SDS-PAGE through a 14% gel in the presence of 1 mM EDTA. Resolution of the three proteins, visualized with Coomassie Brilliant Blue R250, is demonstrated in lane 4, which was loaded with a mixture of the proteins. B) 45Ca2* autoradiography. Following SDS-PAGE, 1.0 frg samples of the three proteins were electrophoretically transferred to nitrocellulose. The resulting replica was then probed with 4sCa2+ (16). exposed to x-ray film for 24 hours at -70” C, and developed, yielding the autoradiograph displayed above. lane 7, avian thymic hormone; lane 2, novel thymic CaBP; lane 3, chicken (muscle) parvalbumin. Apparent molecular masses (in kDa) are indicated between the two panels. 11,700.

The other two proteins,

mobility,

the novel thymic CaBP (lane 2) and the muscle-associated

separable.

Their resolution

however,

is demonstrated

migrate

anomalously.

Although

very similar

parvalbumin

in

(lane 3) are

in lane 4, which had been loaded with a mixture of

the three proteins. The affinity of the novel thymic protein for Ca2+ was demonstrated autoradiography.

The results of a typical experiment

ATH, the muscle parvalbumin, SDS-PAGE, 4sCa2+,

electrophoretically

according

displayed comparable

in intensity

calmodulin

transferred

-- having

illustrates,

to the signals

Small calcium-binding

in Figure 28.

and the putative thymic CaBP protein

to the protocol developed

in Figure 2B clearly

are presented

proteins

to a nitrocellulose by Maruyama

a sequence-derived

et al. (16).

from the two authentic anomalously

molecular

883

weight

Samples

were subjected

membrane,

the signal arising

often migrate

by 4sCa2+ to

then probed

with

As the autoradiograph

from the novel thymic calcium-binding on SDS gels. of 16,800

of

protein

proteins. For example,

(17) -- displays

an

is

Vol.

177,

No.

BIOCHEMICAL

2, 1991

g 20,000

-

‘3 18.000

-.O.o

ii 16.000 II: cl 14,000 + ; 12.000

-

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

-

d I 10.000

-‘--;-i

I

I 40

,I

I 41

I I I I I I I 42 43 44 45 ELUTION VOLUME

I

I 46 (ML)

I

I 47

I

I 46

I

I 49

I

Figure 3. conditions.

Estimation of molecular weight by gel-filtration under denaturing 0.3 ml sample of the novel thymic CaBP (0.5 mg) in 6 M urea, 25 mM HEPES, pH 7.4,l mM EDTA, was loaded onto a 1.5 x 45 cm column of Sephadex G-100 that had been equilibrated with the same solution. The sample also contained 0.5 mg of each of the following proteins, for purposes of calibration: lactoglobulin (18,400), skeletal troponin C (17,800),

lysozyme (14,400), and ATH (11,700). Elution was performed at 5 ml/hour, and 0.5 ml fractions were collected. In the figure above, the molecular weights of the standards have been plotted in logarithmic fashion versus the corresponding peak elution volumes, determined by electrophoresis. The arrow denotes the peak elution volume for the novel thymic CaBP.

apparent

molecular

Oncomodulin

weight

of =20,000

(Mr 11,700),

tissue and neoplasms molecular

weight

on SDS-PAGE

the parvalbumin-like

(19-23),

likewise

of 14,000 (24).

brotein

behaves

Thus, the mobilities

on SDS-PAGE

molecular

In order to obtain a more accurate

novel CaBP, it was subjected A sample applied

are not necessarily

to gel-filtration

of the protein -- denatured

to a gel-filtration

column

greater Stokes radius of the random-coil G-100

for this determination.

molecular

weight,

the proteins

including

was determined

semi-logarithmic

examined

its interaction

properties,

(25,26).

generally

accompanied

reduced

collisional

binding

also

from adjacent

affords

lanthanide

quenching

aromatic

the use of Sephadex

increase

for

of known

The elution volume for each of

of the column fractions.

a molecular

Figure 3 is a

for the four standards.

weight of roughly 11,700. protein,

we then

ions are widely used probes of calcium-binding or Tb3+ by a calcium-binding in luminescence.

of the excited lanthanide

opportunity

conditions.

Similar to Ca2+ in size and coordination

of either E$+

by a dramatic

weight of the

The substantially

several proteins

weight vs. the peak elution volume

with Tb3+ and Eu3+.

an apparent

6 M urea and 1 mM EDTA -- was

that the thymic protein was a small calcium-binding

Sequestration

the

under denaturing

of the column.

with ATH, indicating

placental

of their true

estimate of the molecular

form of the protein necessitated

analysis

displaying

reflections

with the same solution.

by electrophoretic

these luminescent

systems

accurate

in buffer containing

ATH, for calibration

plot of molecular

in mammalian

of the novel thymic CaBP and chicken

The CaBP sample also contained

The novel thymic CaBP co-eluted Having established

expressed

chromatography

equilibrated

of EDTA (e.g.,18).

as a larger protein,

muscle parvalbumin weights.

in the presence

efficient

excitation

amino acid residues. 884

This behavior

ions by water. of the

protein

bound

is

results from

In the case of Tb3+, ion

via

energy

transfer

Vol. 177, No. 2, 1991

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

pH 6.5

o-,

0 4

, 0

, 0.5

,

, , , , , I.0 I5 20 MOLAR EQUIVALENTS

,

, , , 25 3.0 OF Tb3+

,

,35 WAvELENGw

(ANGSTROMS)

Fiaure 4. Titration of the novel CaBP with Tb3* ion. Aliquots of Tb3+ were added to a 50 uM solution of the novel thymic CaBP, in 0.15 M NaCI, 0.025 M MES, pH 6.5, and the Tbs+ luminescence (at 545 nm) was measured after each addition. The excitation wavelength was 260 nm. In the figure above, the signal intensity has been plotted versus the Tb3+: CaBP ratio. Figure 5. Ed+ luminescence spectrum of the novel thymic CaBP. A) Two molar equivalents of Eus+ were added to a 50 pM sample of thymic CaBP, in 0.15 M NaCI, 0.025 M MES, pH 6.5, and the 7Fo+sDo excitation spectrum was obtained. B) Following acquisition of the spectrum shown in panel A. the sample was adjusted to pH 9.0 with 1 M NaOH, and the spectrum was reacquired. Spectrum B was acquired at an instrumental sensitivity three times greater than that used for spectrum A.

When a sample of the novel thymic CaBP was titrated with Tbs+, the luminescence increased abruptly

linearly until two equivalents leveled

irradiating

off (Figure

the sample

phenylalanyl

with Tba+ ion.

the linear increase

indicates

that the lanthanide

typically

display

present, only

(e.g.,

it is sufficient

transition

to note

one, peak in the 7Fo-tsDo

resolved

doublet

centered

spectrum

14,15,30-35)

near

that

indirectly,

spectrum.

observed

during

for the trivalent

ions (27-29).

of the novel CaBP is displayed

the titration ion-binding

binding

site

in a multi-site

elsewhere protein

Below pH 7.0, parvalbumins

A, reflecting

885

the

contributions

from

sites

in Figure 5. The

has been used to study numerous in detail

with

by Ca2+ at the start

Parvalbumin

-- which

each

sites, equivalent

the bound Ca2+.

-- are described

5795

of two ion-binding

in Tb a+ luminescence

preference

excitation

of this electronic proteins

ions were excited

Since both sites were occupied

readily displaced

a much greater

The Eu3+ 7Fo+sDo

binding

the bound

The data suggest the presence

respect to their interaction

properties

In this experiment,

the signal

at 260 nm and relying on energy transfer to Tba+ from nearby

residues.

of the experiment,

4).

of the ion had been added, whereupon

(25). affords

display the

two

calciumFor the one,

and

a partially ion-binding

Vol.

BIOCHEMICAL

177, No. 2, 1991

sites (15,36). broader

As the pH is raised, the spectrum

spectrum

completely

centered

understood

examined

to date.

(37), the phenomenon

Comparabie

The 7Fc+sDo

behavior

unpublished

excitation

Although

is not observed

is not

with all parvalbumins

with calmodulin

or troponin

C (M.T.

observations).

spectra of the novel thymic CaBP at pH 6.5 and pH 9.0 are exhibits a peak near 5795 li and a shoulder

5792 A. At higher pH, this doublet gives way to a much broader

member

by a

the basis for this behavior

has been observed

in Figure 5. The low-pH spectrum

The appearance

RESEARCH COMMUNICATIONS

arising from the CD site is replaced

near 5784 A (14,37).

Henzl and E.R. Birnbaum,

presented

AND BIOPHYSICAL

and pH-dependence

of the parvalbumin

feature centered

at

near 5785 A.

of the spectra clearly identify the novel thymic CaBP as a

family. DISCUSSION

Fish and amphibians (e.g., 38,39).

Mammals,

certain non-muscle (lo),

express

multiple

isoforms

of parvalbumin

on the other hand, produce

tissues as well (5).

but they also express

a distinct

expression

species.

have been no reports of a comparable

There

of a thymus-specific

against ATH fail to cross-react

its reported

ability

parvalbumin

has been labeled

protein

suggests

to stimulate

other physiological We herein distinct detected,

T-lymphocyte

transfer

weight

Whether

its expression

immunomodulatory is clear:

The emerging

muscle isoform complete

spectrum

lanthanide

pattern

Because

parvalbumin,

may serve

(absence

following

of the three proteins

on the basis of its and high

properties.

it likewise

investigation.

expression

-- involving

and without promises

possesses

This much, however,

in the chicken

-- is intriguing

and their functions

Kretsinger, R.H. (1980) CRC Crit. Rev. Biochem. 8, 119-174. Wnuk, W., Cox, J.A., and Stein, E.A. (1982) Calcium Cell Function Gillis, J.M. (1985) Biochim. Biophys. Acta 811, 97-145. 886

and

in thymus tissue than avian thymic hormone. awaiting

isoforms

was originally

SDS-PAGE

of tryptophan

confined to the thymus and whether of parvalbumin

electrophoretically

The protein

precedent.

to be interesting.

REFERENCES 1. 2. 3.

of

the thymus-specific

as a parvalbumin

luminescence

are two questions

and two extramuscular

description

a second

parvalbumin.

is much less abundant

is likewise

capability

thymic tissue, and (40).

Ca2+ ion buffers, pan/albumins

It is classified

UV absorption

PHE: TYR ratio), and characteristic The novel parvalbumin

to avian

The potent effector capacity of this

of ATH, by 45Ca2+ autoradiography

to nitrocellulose.

(=11,500),

to be confined

organisms.

report that the avian thymus harbors

during purifications

(40-42),

(ATH).

serving as cytoplasmic

roles in certain

At the present

thymic extracts

maturation

in

a single muscle isoform

(10,ll).

appears

muscles

is expressed

protein in mammalian

with mammalian

from both ATH and the muscle-associated

electrophoretic molecular

parvalbumin

avian thymic hormone

that, besides

birds express

in the thymus

time, high-level antibodies

a single isoform, which

Like mammals, parvalbumin

in their skeletal

2, 243-278.

a single The

Vol.

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21 . 22. 23.

177,

No.

2, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Celia, M.R., and Heizmann, C.W. (1981) Nature (London) 293, 300-302. Heizmann, C.W. (1988) in Calcium and Calcium Binding Proteins (Gerday, Ch., Gilles, R., and Bolis, L., eds), pp. 93-101, Springer-Verlag, Berlin. Heizmann, C.W., Rohrenbeck, J., and Kamphuis, W. (1989) in Calcium Binding Proteins in Normal and Transformed Cells (Pochet, Ft., Lawson, D.E., and Heizmann, C.W., eds), pp. 57-76, PLenum Publishing Corp., New York Brewer, J.M., Wunderlich, J.K., Kim, D.-H., Carr, M.Y., Beach, G.G., and Ragland, W.L. (1989) Biochem. Biophys. Res. Commun. 160, 11551160. Brewer, J.M., Wunderlich, J.K., and Ragland, W.L. (1990) Biochimie 72, 653-660. Palmisano, W.A., and Henzl, M.T. (1991) Arch. Biochem. Biophys. 285, 21 l-220. Strehler, E.E., Eppenberger, H.M., and Heizmann, C.W. (1977) FEBS Leti. 78, 127-133. Palmisano, W.A., and Henzl, M.T. (1991) Biochem. Biophys. Res. Commun. 176, 328-334. Serda, R.E., and Henzl, M.T. (1991) J. Biol. Chem. 266, 7291-7299. Laemmli, U.K. (1970) Nature 227, 680-685. Henzl, M.T., McCubbin, W.D., Kay, CM., and Birnbaum, E.R. (1985) J. Biol. Chem. 260, 8447-8455. Henzl, M.T., and Birnbaum, E.R. (1988) J. Biol. Chem. 263, 10674-l 0680. Maruyama, K., Mikawa, T., and Ebashi, S. (1984) J. Biochem. 95, 51 l-51 9. Klee, C.B., and Vanaman, T.C. (1982) Adv. Protein Chem. 35, 213-321. Putkey, J.A., Slaughter, G.R., and Means, A.R. (1985) J. Biol. Chem. 260, 4704-4712. MacManus, J.P., and Whitfield, J.F. (1983) in Calcium and Cell function (Anghileri, L.F., and Tuffet-Anghileri, A.M., eds), vol. 4, pp. 411-440, Academic Press, Orlando, FL. Brewer, L.M., and MacManus, J.P. (1985) Dev. Biol. 112, 49-58. Brewer, L.M., and MacManus, J.P. (1987) Placenta 8, 351-363. Gillen, M.F., Brewer, L.M., and MacManus, J.P. (1988) Cancer Lett. 40, 151-160. Sommer, E.W., Blum, J.K., Berger, M.C., and Berchtold, M.W. (1989) FEBS Lett. 257, 307-310.

24. Clayshulte, T.C., Taylor, D.F., and Henzl, M.T. (1990) J. Biol. Chem. 265, 1800-1805. 25. Horrocks, W. dew., Jr., and Sudnick, D.R. (1981) Act. Chem. Res. 14, 384-392. 26. Martin, R.B. (1983) in Calcium in Biology (Spiro, T.G., ed) pp. 235-270, John Wiley & Sons, New York. 27. Corson, D.C., Williams, T.C., and Sykes, B.D. (1983) Biochemistry 22, 5882-5889. 28. Cave, A., Daures, M.-F., and Parello, J. (1979) Biochimie 61, 755-765. 29. Moeschler, J.J., Schaer, J.-J., and Cox, J.A. (1980) fur. J. Biochem. 111, 73-78. 30. Horrocks, W. dew., Jr., and Collier, W.E. (1981) J. Am. Chem. Sot. 103, 2856-2862. 31 . Snyder, A.P., Sudnick, D.R., Arkle, V.K., and Horrocks, W. dew., Jr. (1981) Biochemistry 20, 3334-3339. 32. Wang, C.-L. A., Leavis, P.C., Horrocks, W. dew., Jr., and Gergely, J. (1981) Biochemistry 20, 2439-2444. 33. Horrocks, W. dew., Jr., and Tingey. J.M. (1988) Biochemistry 27, 413-419. 34. Hofmann, T., Eng, S., Lilja, H., Drakenberg, T., Vogel, H.J., and For&n, S. (1988) fur. J. Biochem. 172, 307-313. 35. Palmisano, W.A., Trevitio, C.L., and Henzl, M.T. (1990) J. Biol. Chem. 265, 14450-l

4456.

36. Rhee, M.-J., Sudnick, D.R., Arkle, V.K., and Horrocks, W. dew., Jr. (1981) Biochemistry 20, 3328-3334. 37. Trevino, C.L., Palmisano, W.A., and Henzl, M.T. (1990) J. Biol. Chem. 265, 9694-9770. 38. Pechere, J.-F., Demaille, J., and Capony, J.-P. (1971) Biochim. Biophys. Acta 236, 391-408. 39. Simonides, W.S., and van Hardeveld, C. (1989) Biochim. Biophys. Acfa 998, 137-144. 40. Murthy, K.K., Pace, J.L., Barger, B.O., Dawe, E.L., and Ragland, W.L. (1984) Thymus 6, 43-55. 41. Murthy, K.K., and Ragland, W.L. (1984) in Chemical Regulation of Immunity in Veterinary Medicine, pp. 481-491, Alan R. Liss, Inc., New York. 42. Murthy, K.K., Beach, F.G., and Ragland, W.L. (1984) in Thymic Hormones and Lymphokines (Goldstein, A.L., ed), pp. 375-382, Plenum Press, New York. 887