Distribution of thymosin β4 in vertebrate classes

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 221, No. 2, March, pp. 570-576, 1983

Distribution SUSAN

of Thymosin

p4 in Vertebrate

ERICKSON-VIITANEN, PAOLO NATALINI,l

Roche

Institute

of Molecular Received

AND Biology,

November

Classes

SILVER10 RUGGIERI, B. L. HORECKER Nutleg,

New

Jersey

07110

2, 1982

A peptide containing 43 amino acid residues, rich in glutamic acid and lysine, was originally isolated from calf thymus and designated thymosin & [T. L. K. Low, S. -K. Hu, and A. L. Goldstein (1981) Proc. Nat. Acad. Sti USA 78, 116%11661. However, thymosin p4 was also shown to be present in other tissues of rats and mice, with highest concentrations in spleen and peritoneal macrophages [E. Hannappel, G.-J. Xu, J. Morgan, J. Hempstead, and B. L. Horecker (1982), Proc Nat. Acad. Sci. USA 79,2172-21751. We have now identified the same peptide in tissues of other mammalian species and other vertebrate classes, including birds and amphibia. Exceptions are the rabbit and bony fish, where thymosin p4 is replaced by different peptides, similar in size and in amino acid sequence. None of these peptides was detected in several invertebrates or in the protozoan, Tetrahymena pyrifin-mis. In subcellular fractionation of rat spleen, thymosin p4 was recovered in the cytosol.

We have previously reported that thymosin &, originally isolated from a calf thymus preparation (l), is present in most tissues of rats and mice (2) and also that it is synthesized by cultured peritoneal macrophages and adherent spleen cells from these species (3). In a search for clues to the biological function of this peptide, previously considered to be a thymic hormone (1, 4), we have studied its distribution in the animal kingdom. We now report that thymosin & is a major peptide in human spleen and in several human cell lines, as well as in tissues of cat, chicken, and the toad, Xmopus luevis. Rabbit spleen and liver were found to contain a closely related peptide, differing from thymosin & in only a single amino acid residue. In spleen and liver from a species of bony fish, thymosin /3* is replaced by a closely homologous peptide, whose complete structure remains to be established. In mam1 Present address: Laboratorio plicata, Universita Degli Studi Camerino (MC), Italy.

di Biochimica di Camerino.

0003-9361/33/040570-07$03.00/O Copyright All righb

8 1983 by Academic Press, Inc. of -epmduetion in any form mewed.

malian tissues, but not in tissues from other vertebrate forms, smaller quantities of a different peptide, also closely related to thymosin &, are present.

EXPERIMENTAL

PROCEDURES

These are described in the miniprint supplement or in the tables and figure legends. RESULTS

To minimize the possibility of proteolytic modification, the extracts were prepared from tissues frozen in liquid nitrogen immediately after their removal from the animals and extracted, while still frozen, with 6 M guanidine 0HCl in a Polytron homogenizer (Brinkmann). In experiments with cultured cell lines the cell pellets were resuspended in 6 M guanidine. HCl immediately after harvesting. The extracts were desalted and analyzed by HPLC as described (2). In extracts of human, frog, or fish spleen and of chicken

Ap62032

570

DISTRIBUTION

Human

spleen

OF

THYMOSIN

&

Chicken

(O.&d

IN

VERTEBRATES

bursa

of Fabricius

571

(0.6Q)

1

g '4 P

: 6 L A

,--

-----I

I-I-----.

:

Frog

spleen

‘ish

(0.16g)

spleen

/

(0.42g)

TIME (m!n)

FIG. 1. HPLC analysis of peptides in extracts of human, frog, and fish spleen and the bursus of Fabricius of chicken. Frozen tissues or cell pellets were extracted with 6 M guanidinemHC1 and peptides were concentrated by absorption onto two Waters Sep-pak Cls cartridges in series (2). Elution from the cartridges was with 1 M formic acid/O.2 M pyridine/20% n-propanol. Automated HPLC analysis was carried out using an Ultrasphere ODS Cl8 column (4.6 X 250 mm) and eluting with 1 M formic acid/O.2 M pyridine, pH 2.8, and stepwise increments of n-propanol as indicated (- - -), at a flow rate of 0.55 ml/min. The quantities of tissue extracted were: human spleen, 1.2 g; frog spleen, 0.16 g; fish spleen, 0.42 g; and chicken bursus of Fabricius, 0.57 g. The use of two SepPak cartridges was necessary to obtain a good recovery, which was estimated to be 70%, based on the result obtained on addition of a known quantity of thymosin & to the guanidine . HCl solution before processing.

bursus of Fabricius a major peptide peak was present (Fig. 1) in the position corresponding to that of an authentic sample of thymosin fi4 (2) analyzed on the same column. The chromatographic patterns obtained with the desalted extracts from cat spleen and from two human cell lines, one a promyelocytic leukemia cell line,

designated HL-60 (5), and the other a histolytic lymphoma cell line, designated U937 (6), were very similar (data not shown) to those reported in Fig. 1 for the extract from human spleen. Extracts from chicken spleen and frog liver yielded patterns almost identical to those obtained with extracts from the chicken bursus of Fabri-

ERICKSON-VIITANEN

ET

TABLE AMINO

Residue

ACID

Predicted”

Asp Thr Ser Glu GUY Ala Val Met Ile Leu Phe Tyr LYS His Arg Pro a From known ‘Not analyzed.

4 3 4 11 1 2 0 1 2 2 1 0 9 0 0 3 sequence

COMPOSITION

OF &-LIKE

AL.

I

PEFTIDES

TISSUES AND

IN VERTEBRATE

CELL

LINES

Calf spleen

cat spleen

Rabbit spleen

Human spleen

HL-60 cells

u-937 cells

Chicken spleen

Frog spleen

Fish spleen

4.4 2.4 3.5 11.3 1.3 2.1 0 1.2 1.9 2.3 1.1 0 10.4 0 0 3.5

3.9 2.6 3.9 10.9 1.6 1.7 0.3 1.3 1.9 2.0 1.1 0 8.1 0 0.2 NAb

4.5 2.5 3.4 10.7 2.3 3.3 0.4 1.2 2.0 2.4 1.2 0.2 9.6 0.2 0.9 3.0

5.0 2.5 3.4 10.0 1.4 1.7 0.2 0.8 1.8 2.0 1.0 0.2 8.6 0.2 0.2 3.0

4.4 2.6 3.6 12.1 1.6 2.2 0.3 0.9 1.8 2.0 1.0 0 9.5 0 0 NA

4.0 2.2 3.4 9.8 1.9 2.6 0.5 1.2 1.8 2.2 1.1 0.2 9.2 0.4 0.4 NA

3.5 2.5 3.8 12.0 1.3 2.5 0.3 1.0 1.9 2.2 1.1 0.1 9.8 0 0 NA

3.6 3.2 3.4 10.2 0.8 1.6 0.2 1.0 1.7 1.9 1.0 0 8.7 0 0 3.1

5.0 4.3 3.0 8.4 1.8 2.1 1.1 0 1.0 4.0 1.0 0.2 1.9 0.2 0 3.0

of 6, (1).

cius or frog spleen, respectively. For each extract the major peptide that eluted after the propanol concentration reached 20% had the amino acid composition of thymosin f14(Fig. 2, Table I). This identification was confirmed by analysis of peptide fragments (see miniprint supplement, Tables SI, SII, SIII, and SIV). The COOHterminal sequence was established in each case as Gly-Glu-SerOH by digestion with carboxypeptidase Y (7). No amino acids were detected in one-step Edman degradation, indicating that the NH2 termini were blocked. The quantities of thymosin p4 ranged from 0.48 mg/g of protein for calf spleen to 0.77 mg/g of protein for cat spleen (Table II). These quantities compare with the values of 0.55 and 3.8 mg/g protein previously reported (2) for thymosin p4 in rat and mouse spleen, respectively. The values for human cell lines are similar to those previously reported for rat and mouse macrophages (2). The peptide from rabbit spleen and liver that emerged at the position of thymosin /L14(Fig. 3) was similar in amino acid composition to thymosin /3d(Table I) except for the presence of an additional alanine residue. Analysis of fragments derived from

this peptide revealed that of serine at This peptide mosin @$‘“.

(see miniprint supplement) it contained alanine instead the blocked NH2 terminus. will be referred to as thy-

TABLE CONTENT

OF &LIKE TIEEWES

II

PEI-HDES AND CELL

IN VERTEBRATE LINES’

/a/g

Spleen from

wet weight

Calf Human Cat Rabbit* Chicken Frog Fish”

60 68 97 113 83 67 35

mdg protein 0.48 0.54 0.77 0.90 0.66 0.53 0.223

Human cell lines

pg/lO’ cells

I-IL-60 u-937

0.85 1.70

D The values were calculated from amino acid analysis of the peptide eluted from the HPLC column. The protein content of rat spleen was determined as described (2). The same value for protein content (12% of wet weight) was employed to calculate the ratio of peptide to protein tissues from other species. *Content e Content at the elution

of thymosin @$‘* of the homologous

position

peptide

of thymosin

that

8,.

emerged

DISTRIBUTION

OF THYMOSIN

TIME

& IN VERTEBRATES

573

(mid

FIG. 3. HPLC analysis of peptides in extract of rabbit spleen. 0.66g of rabbit spleen was homogenized and guanidinemHC1 extracts were prepared as described in the legend to Fig. 1 (see also Ref. (2)). HPLC analysis was carried out using an Ultrasphere ODS Cl8 column (4.6 X 256 mm) equilibrated with 1 M formic acid/O.2 M pyridine, pH 2.8. A linear gradient of n-propanol (- - -) was programmed using a Beckman Model 420 microprocessor, at a flow rate of 0.65 ml/min. The linear gradient resulted in earlier elution of thymosin 8, than the stepwise elution employed in Fig. 1. The peptide in peak X appears to be identical to the second peptide from human spleen. The amino acid composition of peptide Y (data not shown) suggests that it is not homologous with thymosin fl,.

The corresponding peptide from the spleen of a species of bony fish (Astrmotw ocellutus) differed from thymosin IB4 with respect to the quantities of several amino acids, and also in containing valine, but no methionine (Table I). Preliminary results (unpublished) indicate that this peptide is closely homologous to thymosin &, but its structure has not yet been determined. All of the mammalian tissues and cell lines analyzed, including rabbit spleen and liver, yielded a second peptide that emerged approximately 3-5 min later than thymosin & (see Fig. 1, human spleen). Generally, the quantity of this peptide was lo30% that of thymosin & except in the rabbit (Fig. 3), where it was present in quantities equal to or greater than those of thymosin fi4. This second peptide appears (unpublished observation) to possess sequences common to both thymosin & and thymosin B9, previously isolated from calf thymus (2,8). In extracts from tissues from lower vertebrate forms this second peptide was not present. Several invertebrate sources (gypsy moth larvae and the common earthworm) were also extracted and analyzed, but no peptide corresponding to thymosin /?4 was

detected. Extracts of Tetrahgmena pyrialso yielded negative results (data not shown). Analysis of subcellular fractions prepared from homogenates of rat spleen showed that fi4 was localized in the cytosol, with negligible quantities recovered in the nuclear, mitochondrial/lysosomal, or microsomal fractions (see miniprint supplement Table SV).

fmis

DISCUSSION

Although the physiological role of these peptides remains unknown, their presence in quantities approaching 100 rg/g of fresh tissue (see Table II) suggests that they fulfill some essential cell function. Thymosin fi4 appears to be the most widely distributed in the vertebrate kingdom and was found to be present in all classes except bony fish, where it is replaced by a homologous peptide. In mammals it is always accompanied by a second, apparently structurally related, peptide. These peptides are present in the cytosol and their large quantities and wide tissue distribution make it unlikely that they function

574

ERICKSON-VIITANEN

as hormones. It is more likely that they contribute to some process that is common to most cells and tissues, possibly as structural elements. REFERENCES 1. Low, T. L. K., Hu, S.-K., AND GOLDSTEIN, A. L. (1981) Proc Nat. Acad. Sci USA 78,1162-1166. 2. HANNAPPEL, E., Xv, G.-J., MORGAN, J., HEMPSTEAD, J., AND HORECKER, B. L. (1982) Proc. Nat. Ad Sci. USA 79,2172-2175. 3. Xv, G.-J., HANNAPPEL, E., MORGAN, J., HEMPSTEAD, J., AND HORECKER, B. L. (1982) Proc. Nat. Acad Sci USA 79,4006-4009.

ET

AL.

4. REBAR, R. W., MIYAKE, A., Low, GOLDSTEIN, A. L., (1981) S&nce

T. L. K., AND 214, 669-671.

5. COLLINS, S. J., GALLO, R. C., AND GALLAGHER, R. E. (1977) Nature (LondonJ 270, 347-349. 6. SUNDERSTRUM, C., AND NILSSON, Can&-r 17, 565-567.

K. (1976)

Int

J.

7. JONES, B. N., PUBO, S., AND STEIN, S. (1981) Liq. Chrmnatogr. 4, 565-586.

J.

8. HANNAPPEL, E., DAVOUST, S., AND HORECKER, B. L. (1982) Proc Nat. Acad. Sci USA 79,17021711. 9. HANNAPPEL, E., DAVOUST, S., AND HORECKER, B. L. (1982) B&hem. Biophys. Res. Commun 104,266-271.

DISTRIBUTION

OF

THYMOSIN

/t$ IN

VERTEBRATES

575

576

ERICKSON-VIITANEN

ET

Sl. 52. 53. 54. s5. 56. 9. SB. $9.

AL.