Human y-trace, a basic microprotein: Amino acid ... - Europe PMC

4 downloads 38 Views 1MB Size Report
Tarr, G. E., Beecher, J. F., Bell, M. & McKean, D. J. (1978). Anal Biochem. 84, 622-627. 19. ... Sternberger, L. A. & Joseph, S. A. (1979) J. Histochem. Cyto- chem.
Proc. NatL Acad. Sci. USA Vol. 79, pp. 3024-3027, May 1982

Medical Sciences

Human y-trace, a basic microprotein: Amino acid sequence and presence in the adenohypophysis (candidate hormone/immunohistochemistry)

ANDERS GRUBB AND HELGE LOFBERG Departments of Clinical Chemistry, Pathology, and Dermatology, University of Lund, Malmo General Hospital, S-214 01 Malm6, Sweden

Communicated by Jan G. Waldenstrbm, January 18, 1982

mented by cyanogen bromide treatment (13) or by tryptic digestion after reversible blocking of amino groups by citraconylation (14). One of the cyanogen bromide fragments (no. 3) was further digested with Staphylococcus aureus V8 protease. On each occasion the fragments were separated by gel chromatography on a column of Sephadex G-50 superfine (1.5 x 190 cm) in 30% (vol/vol) acetic acid. The absorbance at 280 nm, the radioactivity (14C), and the ninhydrin-reactivity ofthe column fractions were measured. All cyanogen bromide fragments and most of the tryptic peptides were pure after the gel filtration, but some tryptic peptides had to be further purified by preparative highvoltage paper electrophoresis (15). Amino acid analysis was performed by standard methods (16) after hydrolysis in 6 M HC1. Automated amino acid sequence analysis by the method of Edman and Begg (17) was carried out on a Beckman 890 C sequencer by using the standard Beckman 1 M Quadrol program. All fragments (50-300 nmol) were run in the presence of Polybrene (18). Phenylthiohydantoin derivatives of amino acids were identified by high-performance liquid chromatography on a 30-cm Waters uBondapak C18 column using stepwise elution with methanol-containing buffers (Waters Associates, internal communication 03-LS-11-76). This detection system identified the phenylthiohydantoin derivatives of hydroxyproline as two peaks at unique positions in the elution diagram. All phenylthiohydantoin derivatives of amino acids were also identified by thin-layer chromatography (19). Carboxyl-terminal amino acid residues were identified after digestion with carboxypeptidase Y (20). Immunohistochemical Procedures. Pituitary glands were taken at operation from a 2.6-kg, 2-year-old adolescent male capuchin monkey, Cebus apella; a 5.9-kg, 13-year-old adult male African green monkey, Cercopithecus aethiops; and, at autopsy 8 hr post mortem, from a 75-year-old man. The specimens were snap-frozen and freeze-dried, vapor-fixed in diethyl pyrocarbonate (21), and vacuum-embedded in Paraplast (Sherwood Medical Industries, St. Louis, MO). Tissue sections, 2,um thick, were examined with the Sternberger peroxidase-antiperoxidase technique (22). A rabbit antiserum against human y-trace was used in the first step ofthe assay. Its production and specificity have been reported (8), and the precipitating titer according to Becker (23) was 0.19 mg of y-trace per ml of

ABSTRACT The amino acid sequence of human V-trace, a basic microprotein without known function, was determined by automated Edman degradations of the carboxynethylated polypeptide chain and of fragments obtained by cyanogen bromide treatment and tryptic digestion after blocking of lysine residues. The single polypeptide chain contained 120 residues, and the calculated Mr was 13,260. A proline residue at position 3 was partly hydroxylated. The presence of y-trace in a significant proportion of the cells in the anterior lobe of simian and human pituitary glands was demonstrated by immunohistochemical procedures with a rabbit antiserum against human V-trace. The tissue localization and amino acid sequence of V-trace indicated that this protein is connected with the peptidergic gastroenteropancreatic neuroendocrine system. Human y-trace is an alkaline, low Mr protein first described in 1961 as a constituent ofnormal cerebrospinal fluid and of urine from patients with renal failure (1-3). The biological role of ytrace is unknown, although the protein has been shown to occur in low concentration not only in cerebrospinal fluid and urine but also in plasma, saliva, and semen (4-6). A sensitive enzyme immunoassay for measurement of y-trace has been described (7). It allowed quantitation of the protein in all human fluids investigated and demonstrated that, in healthy adults, the concentration of y-trace in cerebrospinal fluid is 5.5 times that in plasma. Recent immunohistochemical studies of y-trace have shown it to be present in some human brain cortical neurons, in adrenal medulla, and in the A cells of the pancreatic islets (8-10). Some amino acid sequence similarities also have been found between human glucagon and the amino-terminal part of y-trace (8). Therefore, the question whether y-trace is related to the peptidergic gastroenteropancreatic neuroendocrine system has arisen. This report describes the complete amino acid sequence of y-trace and presents immunohistochemical evidence of the presence of the protein in a large number of cells in the pituitary gland. Experimental details of the work will be

published separately. MATERIALS AND METHODS Isolation of V-Trace. Urine containing about 20 mg of y-trace per liter from patients with renal failure was collected in bottles containing the protease inhibitor benzamidinium chloride and the antimicrobial agent sodium azide. y-Trace was isolated from the pooled urine as described (8). Its purity was checked by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (11) and agarose gel electrophoresis (12). Structural Determination. Isolated y-trace was reduced and carboxymethylated with '4C-labeled reagent in 6 M guanidinium chloride. The carboxymethylated protein was then frag-

antiserum.

RESULTS Primary Structure of Human V-Trace. All cyanogen bromide fragments of y-trace and all tryptic peptides of the citraconylated protein were isolated and used in the determination of the complete amino acid sequence (Fig. 1). The cyanogen bromide fragment 4 was devoid of homoserine and was, therefore, identified as the carboxyl-terminal fragment. The amino acid residues at virtually all positions in the polypeptide chain were identified in at least two different fragments. The proline residue at position 3 was found to be hydroxylated in all three

The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. ยง1734 solely to indicate this fact.

3024

Proc. Natd Acad. Sci. USA 79 (1982)

Medical Sciences: Grubb and L6fberg 10

*

1

3025 20

Ser-Ser-Pro-Gly-Lys-Pro -Pro -Arg-Leu -Val-Gly-Gly-P ro -Met-Asp -Ala-Ser-Val -Glu -GluCNBr 2

CNBr 1 T 2

T i1

t~

-&

-,l

-:i

-..

A

-.-"&

-h

_Iu

--%

--%

-b

v

-h

30

-N

40

Glu-Gly-Val-Arg -Arg-Ala-Leu-Asp-Phe-Ala-Val-Gly-Glu-Tyr-Asn-Lys-Ala-Ser-Asn-AspCNBr 2 T 2 -

1h

-

T 4

T 3 1h

l

l

50

60

Met-Tyr-His-Ser-Arg-Ala-Leu-Gln-Val-Val-Arg-Ala-Arg-Lys-Gln-Ile-Val-Ala-Gly-ValCNBr 3 CNBr 3 S 1

--"&

-"

T 6

T 5

T 4

--%

-%

v

x- 6

"

--,

--%

--%

0

-"

I

v

,

-

.-lb.

K

T 7 ,-1.

70

--6

80

Asn-Tyr-Phe-Leu-Asp -Val-Glu-Leu-Gly-Arg-Thr-Thr-Cys-Thr-Lys-Thr-Gln-Pro -Asn-LeuCNBr 3 -&.~ H

~ _I,b

%-

-

_N.

,

-"

lb

__h

-

,

__%

CNBr 3 S 2

CNBr 3 S 1

T 8

T 7

90

100

Asp-Asn-Cys-Pro-Phe-H is-Asp-Gln-Pro-Hi s-Leu-Lys-Arg-Lys-Ala-Phe-Cys-Ser-Phe-GlnCNBr 3 CNBr 3 S 2 -%.--&. -.& -.%.__,

-,& --..

-%.

-"k.

--Nb

--.bb

%.

--%

--%.

---%.

.-Mb

T 9

T 8 --bb

--Nb

--.%b

--

A&

--.%b

--%.--

---2bb

--lb

---116

--,&

--.lb

--.bb

---Nb

110

--Ab

120

I le-Tyr-Ala-Val-Pro-Ser-Gln-Gly-Thr-Met-Thr-Leu-Ser-Lys-Ser-Thr-Cys-Gln-Asp-Ala CNBr 3

CNBr 4

CNBr 3 S 2 T 9

FIG. 1. The complete amino acid sequence of human y-trace and the fragments used in the deduction of the sequence. CNBr, cyanogen bromide fragment; T, peptides isolated from a tryptic digest of citraconylated y-trace; CNBr 3 S, peptides obtained after digestion of cyanogen bromide fragment 3 with Staphylococcus aureus V8 protease; -, amino acid residue identified by automated Edman degradation; -, amino acid released by carboxypeptidase Y digestion; *, partly hydroxylated residue.

investigated preparations of pure y-trace. Hydroxyproline was identified at position 3, both in the uncleaved polypeptide chain and in the corresponding tryptic and cyanogen bromide fragments thereof. It was demonstrated in all cases both by amino acid analyses and by high-performance liquid chromatography of the corresponding phenylthiohydantoin derivatives. The hydroxylation degree was about 50% but varied slightly in three investigated preparations of y-trace. The polypeptide chain contained 120 residues and had a calculated Mr of 13,260. When the sequence of y-trace was aligned with known sequences of hormones belonging to the gastroenteropancreatic

neuroendocrine system, two similarities were found. Both concerned the 35-residue-long amino-terminal part of y-trace, which showed sequence similarities with human corticotropin (comprising 8 of its 39 residues) and with glucagon (comprising 7 of its 29 residues) (Fig. 2). Localization of y-Trace in the Pituitary Gland. Biopsy specimens containing adenohypophyseal tissue were identified by immunohistochemical techniques with antisera against human somatotropin and human corticotropin. In sections from simian and human adenohypophyses, there was immunostaining of ytrace in the cytoplasm of numerous epithelial cells, when native

Proc. Nad Acad. Sci. USA 79 (1982)

Medical Sciences: Grubb and L6fberg

3026

Corticotropin

Y-Trace

Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val Gly-Lys -Lys-Arg 5 1

-Pro -Val-

10io

Ser-Ser-Prol Gly-Lys Pro-Pro,-Argy-Leu-IVaj His-Ser-Gln-

Glucagon

Corticotropin

Lys-Val-Tyr-Pro Asp-Ala -Gly-Glu-Asp-Gln-Ser-Ala-Glu-Ala-Phe-Pro- Leu -Glu 11-

[-Trace

Gly-Gly-Pro-Met

Glucagon

20

25

Ser-Val-Glu-Glu-Glu-Gly-Val

Arg-Arg-Ala

Phe 30

Asp-Phe

Ala-

Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Lsu-Asp-SerlArg-Arg-Ala rGlnfAsp-Phe

Val-

31

[-Trace

~~~~15 Asp-Ala

Leu

40

-35-

Val-Gly-Glu-Tyr- Asnf-Lys-Ala-Ser-Asn-Asp-

Gln-Trp-Leu-MettrAr -Thr

Glucagon

FIG. 2. The amino acid sequence of human y-trace compared to the sequences of human glucagon (24) and human corticotropin (25). Identical residues of the molecules are shown in boxes.

antiserum against y-trace, specific antibodies against y-trace, or Clq-depleted native antiserum against y-trace was used (Fig. 3). No immunostaining was obtained when serum from a nonimmunized rabbit was used. The histochemical immunoreactivity ofthe antiserum against y-trace disappeared after passage i

i.

f

of the antiserum through a column containing insolubilized ytrace or after absorption of the antiserum with pure soluble ytrace. The immunoreactive end point of serial dilutions of the antiserum was 1:1,600. There was no decrease in the immunoreactivity against y-trace of the antiserum after overnight in-

.04

"w

t. 6

4

I

L;S *~s -

4.

4,_4 |vs.w.a S,....

k I

4..

io-

A.

N V

J''^A

A.4

:.

vIr,9

Ian.

FIG. 3. High-power photomicrographs, showing peroxidase-antiperoxidase immnunostaining of y-trace in the pituitary gland of a 13-year-old male African green monkey (A) and in a 2-year-old male capuchin monkey (B). -Trace (dark color) is present in the cytoplasm of numerous adenohypophyseal cells. Both large cells rich in cytoplasm (A) and small-sized cells (B) contain y-trace. (A, x575; B, x575.)

Medical Sciences: Grubb and lbfberg

Proc. Nati Acad. Sci. USA 79 (1982)

cubation with human somatotropin, human prolactin, bovine thyrotropin, porcine corticotropin, synthetic corticotropin-(1-28), and porcine glucagon in molar amounts corresponding to about 20 times the precipitating titer of the antiserum. DISCUSSION

The complete amino acid sequence of y-trace presented here agrees with two earlier published sequences of the amino-terminal part of the protein (8, 26). The amino acid composition calculated from the sequence also conforms to the one obtained after acid hydrolysis (Table 1). The amino acid sequence of ytrace contains three pairs (or triplets) of basic amino acid residues (positions 24 and 25, 53 and 54, and 92-94) which might be of considerable interest because it is known that several hypophyseal hormones are produced by proteolytic cleavage of prohormones at such pairs of basic amino acid residues (27). Recent immunohistochemical studies have demonstrated the presence of y-trace in chromaffin cells of adrenal medulla, A cells of pancreatic islets, and in a number of brain cortical neurons (8-10). The results of this work show that y-trace also is localized in a large proportion of the cells of the adenohypophysis. These localization studies, taken together, point to a tissue distribution of y-trace, suggesting that it might be a part of the peptidergic gastroenteropancreatic neuroendocrine system. A connection between y-trace and the peptidergic neuroendocrine system is also indicated by the amino acid sequence similarities between human corticotropin, glucagon, and ytrace. Table 1. Amino acid composition of human y-trace Constituent Half-cystine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine

Leucine

Tyrosine Phenylalanine Lysine Histidine Arginine *

Composition of acid hydrolysate* 3.7 12.0 7.4 8.1 11.9 8.2 9.3 10.0

Composition calculated from sequence 4 12 7 9 12 8 8

10.1 2.4

2.1 8.2 3.8 5.1 6.9 2.9 8.0

From L6fberg et al. (8); recalculated to 120 residues.

10 10 3 2 8 4 5 7 3 8

3027

The sklflfull technical assistance of Veronica Grimsberg, Leif Hansson, and Roland Nilsson is gratefully acknowledged. The biopsy specimens were obtained in collaboration with Dr. Lars-Goran Str6mblad, Department of Neurosurgery, University Hospital, Lund, and Dr. Sven-Olle Olsson, AB Ferrosan, Malmo, Sweden. This investigation was supported by grants from the Swedish Medical Research Council (Project B 82-13X-05196-05A); the Medical Faculty, University of Lund; Konung Gustaf V:s 80-Arsfond; the Swedish Society of Medical Sciences; and Neurologiskt Handikappades Riksforbund. The sequenator was obtained through a grant from the Wallenberg Foundation.

1. Butler, E. A. & Flynn, F. V. (1961) . Clin. Pathol 14, 172-178. 2. Clausen, J. (1961) Proc. Soc. Exp. BioL Med. 107, 170-172. 3. MacPherson, C. F. C. & Cosgrove, J. R. (1961) Can.J. Biochem. 39, 1567-1574. 4. Cejka, J. & Fleischmann, L. E. (1973) Arch. Biochem. Biophys. 157, 168-176. 5. Colle, A., Guinet, R., Leclercq, M. & Manuel, Y. (1976) Clin. Chim. Acta 67, 93-97. 6. Hochwald, G. M. & Thorbecke, G. J. (1962) Proc. Soc. Exp. BioL Med. 109, 91-95. 7. L6fberg, H. & Grubb, A. 0. (1979) Scand. J. Clin. Lab. Invest. 39,-619-626. 8. LUfberg, H., Grubb, A. 0. & Brun, A. (1981) Biomed. Res. 2, 298-306. 9. L6fberg, H., Nilsson, K. E., Str6mblad, L.-G., Lasson, A. & Olsson, S.-0. (1982) Acta Endocrinol (Copenhagen), in press. 10. LUfberg, H., Stromblad, L.-G., Grubb, A. 0. & Olsson, S.-O. (1981) Biomed. Res. 2, 527-535. 11. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 12. Johansson, B. G. (1972) Scand. J. Clin. Lab. Invest 29, Suppl. 124, 7-19. 13. Steers, E., Jr., Craven, G. R., Anfinsen, C. B. & Bethune, J. L. (1965)J. BioL Chem. 240, 2478-2484. 14. Dixon, H. B. F. & Perham, R. N. (1968) Biochem. J. 109, 312-314. 15. Mendez, E. & Lai, C. Y. (1975) Anal Biochem. 65, 281-292. 16. Spackman, D. H., Stein, W. H. & Moore, S. (1958) AnaL Chem. 30, 1190-1206. 17. Edman, P. & Begg, G. (1967) Eur. J. Biochem. 1, 80-90. 18. Tarr, G. E., Beecher, J. F., Bell, M. & McKean, D. J. (1978) Anal Biochem. 84, 622-627. 19. Jeppsson, J.-O. & Sj6quist, J. (1967) AnaL Biochem. 18, 264-269. 20. Hayashi, R. (1977) Methods Enzymol 47, 84-93. 21. Pearse, A. G. E. & Polak, J. M. (1975).Histochem.J. 7, 179-186. 22. Sternberger, L. A. & Joseph, S. A. (1979) J. Histochem. Cytochem. 27, 1424-1429. 23. Becker, W. (1969) Immunochemistry 6, 539-546. 24. Thomsen, J., Kristiansen, K., Brunfeldt, K. & Sundby, F. (1972)

FEBS Lett. 21, 315-319.

25. Lee, T. H., Lerner, A. B. & Buettner-Janusch, V. (1961)J. BioL Chem. 236, 2970-2974. 26. Tonnelle, C., Colle, A., Fougereau, M. & Manuel, Y. (1979) Biochen. Biophys. Res. Commun. 86, 613-619. 27. Nakanishi, S., Inoue, A., Kita, T., Nakamura, M., Chang, A. C. Y., Cohen, S. N. & Numa, S. (1979) Nature (London) 278, 423-427.