Comparative Study of Primates' Transcortin: Immunoreactivity and Steroid-Binding Activity

Endocrinol. Jzpon. 1986, 33 (2), 143-150

Comparative Study of Primates' Transcortin: Immunoreactivity and Steroid-binding Activity

KAORI SUEDA*, HISAO SEO AND NOBUO MATSUI

The Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464 * The Department of Home-economics, Nagoya Women's University, Takamiya-cho, Tenpaku-ku, Nagoya, 468

Abstract

To investigate the phylogenic aspect of transcortin (corticosteroid-binding globulin, CBG), the immunoreactivity of transcortin with anti-human transcortin antiserum was studied in primates. The anti-human transcortin antibody was recognized by plasma proteins obtained from Catarrhini, taxonomi- cally the most evolved monkey group. The immunoreactivity was not observed in plasma obtained from Platyrrhini and Prosimiae, classified as less evolved monkey groups than Catarrhini. Though comparison of immunoreactivity among different classes of Catarrhini was difficult because of non-parallelism of their displacement curves, displacement of 125I-labelled human transcortin from the antiserum by 1: 10 and 1: 100 diluted plasma was highest in human followed by Pongidae, Cercopithecoidea. The immunoreactivity of thyroxine-binding globulin (TBG) with anti-human TBG antiserum was also examined. The anti-human TBG antibody was only recognized in plasma from Pan (anthropoid ape) among Pongidae, highly evolved monkeys among Catarrhini. The existence of immunoreactive transcortin and TBG to respective human protein antibody in the highly evolved ape agreed well with the cladogenetic division of primate species delineated by Goodman and Moore (1971). Cortisol-binding activity of transcortin was detected in all monkeys except three, tafted capuchin monkey, night monkey and cotton-headed tamarin, which belong to Platyrrhini. The absence of cortisol-binding activity in these animals might be attributed to high levels of endogenous cortisol and low cortisol-binding capacity of transcortin. It is speculated that the structure of the immunoreactive site in transcortin could be modified by evolution without affecting the biologically important site, the site for cortisol binding.

The first appearance of vertebrates dates billions years (Burlingame et al., 1965). back to the paleozoic era, about 500 to 600 Seal and Doe(1963) determined corticosteroid- million years ago, and that of biogenetic binding activity in the plasma of a great steroids has been traced back more than 2 number of different species and suggested that the basic phenomenon of serum protein Received July 12, 1985 binding of corticosteroids appeared early in Endocrinol. Japon. 144 SUEDA et al. April 1986 the evolutional history of vertebrates and 3. Examination of cortisol-binding activity of that high structural specificity of the protein animal transcortin which offers strong binding forces for the Five ƒÊ1 of plasma sample was incubated with 0.1ƒÊ1(1 ƒÊCi/4ng) of 3H-cortisol at 37•Ž for 30 corticosteroid hormones must have been min, then 2 ƒÊ1 of the mixture was applied on conserved through evolution. However, mole- agarose gel and electrophoresed for 45 min at

cular evolution of transcortin in the aspect 15 mA in barbital buffer pH 8.5. After electro- of immunochemical properties has not been phoresis, the gel was dried and autoradiographed studied yet. with Kodak X-Omat S film for 2 weeks at -80℃ The relative position of radioactivity In the present report, the immunological reactivity with anti-human transcortin anti- on the gel was connrmed by staining the gel with Amido black 10B. body and the steroid-binding activity of non-human primate's plasma were studied. 4. Immunoreactivity of non-human primate trans- Further, immunoreactivity with anti-human cortin and TBG with anti-human transcortin thyroxine-binding globulin (TBG), another or TBG antiserum hormone-binding protein in the plasma, was The immunoreactivity of non-human primate also determined in the same plasma. transcortin with anti-human transcortin antiserum was assessed by competitive binding between radiolabeled human transcortin and unlabeled

animal transcortin to the antiserum (Sueda et al., Materials and Methods 1983). In this assay system, the least detectable amount of human transcortin was 1ng/0.1 ml.

1. Material A mixture consisting of 0.1 ml diluted plasma 3H-cortisol (88ƒÊCi/mmole) , Na125I (174 mCi/ sample, 0.1 ml 125I-transcortin solution (10,000 μg)were obtained from Amersham International cpm/1.3ng) and 0.1 ml antiserum solution (dilu- plc. (Buckinghamshire, England) and agarose tion of 1: 100,000) was incubated for 2 days at plates (Universal electrophoresis filmR) were ob- 4s•Ž. After the incubation, 50 ƒÊ1 of normal tained from Corning Co. (CA., U.S.A.). Trans- rabbit serum was added and antibody-bound cortin was purified from pooled human serum transcortin was precipitated by adding 1ml of and antiserum to human transcortin was raised 25•“ polyethylene glycol solution in 0.1M borate in a rabbit (Sueda et al., 1983). Purified human buffer pH 8.6. Then the mixture was centrifuged

TBG and anti-TBG antiserum were gifts from at 3,000 rpm for 15 min and radioactivity of the

Dr. S. Refetoff (Chicago University, U.S.A.). precipitate was counted with a well-type NaI (T1) Iodination of transcortin and TBG was carried scintillation counter. This determination was per- out according to the Chloramine-T method formed at three different concentrations of plasma

(Greenwood and Hunter, 1963). All other re- using 1: 10, 1: 100 and 1: 1,000 diluted plasma. agents were purchased from Wako Pure Chemical Phosphate buffered saline pH 7.4 containing 1% Institute Ltd. (Osaka, Japan), and were of ana- bovine serum albumin was utilized to dilute lytical grade. plasma and the antiserum. The immunoreactivity of primate TBG with anti-human TBG antiserum

2. Blood samples was also examined in the same manner as that Thirty seven plasma samples from 20 non- of transcortin, using three concentrations of human primate species were generously supplied plasma, 1:50, 1:100 and 1:200 dilution. The by Dr. M. Minezawa at The Primate Institute least detectable amount was 0.3ng TBG/0.1ml. of Kyoto University (Inuyama, Japan). All Immunoreactivity of the sample is thought to primates examined are listed in evolutional order be present when radiolabeled ligand-displacing in Table 1. Plasma of goat, dog, rabbit, musk activity . of the sample was dose dependent and rabbit, rat, guinea pig and mouse were obtained the most diluted plasma sample exhibited signifi- atour institute, and those of cow and pork cant displacement. were provided by Dr. K. Sato (Nagoya Univer- sity, School of Agriculture, Nagoya, Japan).

Samples were kept frozen (-20•Ž) until use. Vol.33, No.2 EVOLUTIONAL STUDY OF TRANSCORTIN 145

or 2 monkeys of Prosimiae (Fig. 2). On the Results other hand, the immunoreactivity of primate's plasma with anti-human TBG antiserum was 3H-cortisol incubated with plasma was positive in only 3 Pongidae, i.e., chimpan- separated into three major fractions by agar- zee, gorilla and orang-utan, as shown in gel electrophoresis, and a typical result of Fig. 3. Other Pongidae such as siamany autoradiography is shown in Fig. 1. In gibbon, dark-handed gibbon and white- addition to a free cortisol fraction (origin), handed gibbon gave negative results, and two protein-bound cortisol fractions were no other monkeys showed positive immuno- recognized. Little radioactivity was accumu- reactivity with anti-human TBG antiserum. lated in the region corresponding to albumin, Mammalian plasma other than that of and a larger amount of radioactivity was primates (cow, fetal cow, goat, pork, dog, observed in a more slowly migrating region rabbit, musk rabbit, rat, guinea pig and than albumin on the gel. The latter is mouse) reacted neither with anti-human considered to be transcortin-bound cortisol. transcortin antiserum nor with anti-human The presence or absence of cortisol-binding TBG antiserum. The results for cortisol- activity thus determined is summarized in binding activity and immunoreactivity were Table 1. Cortisol-binding activity of trans- summarized in Table 1. cortin was detected in all samples examined, except tafted capuchin monkey, night monkey and cotton-headed tamarin. Discussion When the immunoreactivity of primate's plasma transcortin with anti-human trans- In the present study, 3H-cortisol-binding cortin antiserum was analyzed by competitive activity of transcortin was detected in all binding with labeled human transcortin, monkeys except three, tafted capuchin mon- Hominoidea and Cercopithecoidea monkeys key, night monkey and cotton-headed examined gave positive results, as shown in tamarin. All these monkeys belong to the Fig. 2. The immunoreactivity was not re- New World primates showing glucocorticoid cognized in 4 monkeys belong to Platyrrhini resistance with markedly elevated plasma

Fig. 1. Agar-gel electrophoresis of primate's plasma with 3H-cortisol. Abbreviations are as follows: Ori: origin; Tr: transcortin; Al: albumin. Endocrinol. Japon. 146 SUEDA et al. April 1986

Table 1. Transcortin and TBG in Primate

A: Number of Sample. B: Cortisol-binding activity of transcortin. C: Immunoactivity with anti-human transcortin antibody. D: Immunoreactivity with anti-human TBG antibody. glucocorticoid levels (Chrousos et al., 1982; of endogenous cortisol and low cortisol- Yamamoto et al., 1977). In addition, Pugeat binding affinity and/or small cortisol-binding et al. (1984) reported that the cortisol-bind- capacity of their transcortin. ing capacity of plasma transcortin in the The immunoreactivity of transcortin and New World primates was 1/10th to 1/100th TBG in non-human species with anti-human those in the Old World primates and Pro- protein antibodies was compared with that simians, and Yamamoto et al. (1977) reported of human. Immunoreactive transcortin with that the cortisol-binding affinity of trans- anti-human transcortin antiserum was not cortin was low in these species. Therefore, detected in non-primate animals, which is the absence of transcortin-bound 3H-cortisol in agreement with previous data (Muldoon in them might be attributed to high levels and Westphal, 1967; Robinson et al., 1985). Vol.33, No.2 EVOLUTIONAL STUDY OF TRANSCORTIN 147

A B C

D E

Fig. 2. Immunoreactivity with anti-human transcortin antiserum.

Immunoreactivity of serially diluted primate's plasma was tested by competitive binding with 125I-human transcortin. The% 125I-human transcortin bound to the auti-human transcortin antiserum at each dilution is expressed relative to the amount bound in the

absence of human-transcortin (BO). A: plasma sample from Hominidae (human). B: plasma samples from Pongidae • gorilla,

■ siamany gibbon, ▼ orang-utan, *dark-handed gibbon, △ white-handed gibbon, ○ chim- panzee. C: plasmas amples from Cercopithecoidea, □ rhesus monkey, ■ savannah monkey, ▼ bonnet monkey, △ Japanese monkey, ○ patas monkey, ● stump-tailed monkey, ▲

pig-tailed monkey, ▽ gelada baboon. D: Plasma samples ffom Platyrrhini, □ tafted capuchin monkey, ■ cotton-headed tamarin, ▼ common squirrel monkey, △ night monkey.

E: plasma samples from Prosimiae, □ grand tailed galago, ■ slow loris.

Reymore and Kuhn (1983) also reported limited. that the rabbit anti-rat transcortin antiserum In primates, the immunoreactivity of did not exhibit any measurable cross-reacti- plasma transcortin with anti-human trans- vity with plasma obtained from human, cortin antiserum differs according to classes monkey, dog, sheep, guinea pig, while the (Fig. 2). The displacement of 125I-labeled antiserum slightly reacted with mouse serum. human transcortin from anti-human trans- Thus, it might be said that structural homo- cortin antiserum was seen in human, Pon- logy in the protein between species was gidae and Cercopithecoidea. It was not Endocrinol. Japon. 148 SUEDA et al. April 1986

A B

Fig. 3. Immunoreac- tivity with anti- human TBG antiserum. See legend to Fig. 2. A: displacement of human-TBG.B: Plasma sample obtained from Pan in Pongidae.

observed in Platyrrhini or Prosimiae. The decreased immunoreactivity might be attributed to low cross-reactivity due to structural changes. Displacing activity of diluted plasma in Catarrhini (Hominidae, Pongidae and Cercopithecoidea) is summarized in Fig. 2'. Though comparison of their immunoreactivity was difficult as their displacement curves were not parallel, human plasma diluted 1: 10 and 1: 100 most strongly dis- placed the 125I-labeled human transcortin from the antiserum. The displacement at the same dilution of plasma was less in Pongidae and the least in Cercopithecoidea. Robinson et al. (1985) reported that ape transcortin is immunologically identical to human transcortin and that Old World monkey (Hominoidea and Cercopithecoidea) transcortin comprises most, but not all of the human transcortin epitopes. Our results indicate that immunoreactivity of primate plasma might correlate with taxonomic dis- tance, suggesting that the molecular structure of protein changed step by step with evolutional history. TBG is thought to have appeared when Fig. 2' Comparison of displacing activity among Catarrhini. The mean and standard error Ayes evolved to Mammalia (Tanabe el al., 1970). Tanabe et al. (1974, 1977) studied (S.E.) were calculated from the data in Fig. 2. T4-binding protein in primates and reported that TBG occurred in Catarrhini, Platyrrhini and also in common tupai and grand tailed Vol.33, No.2 EVOLUTIONAL STUDY OF TRANSCORTIN 149

Immunoreactivity with anti-human antibody

Transcortin TBG

Fig. 4. Relation between appearance of immunoreactive transcortin and TBG against anti-human protein antibodies and cladogenetic tree (Goodman and Moore, 1971). ●positive in immunoreactivity, ○ negative in immunoreactivity. galago belonging to Prosimiae. Our present first appearance of immunoreactive transco- data showed that immunoreactive TBG with rtin against anti-human transcortin antiserum anti-human TBG antiserum was only ob- was calculated to be about 60 million years served in Pan of Pongidae. Thus, our data ago. The appearance of immunoreactive TBG showed that the structural homology in TBG traced back about 30 million years ago. It was also maintained within a limited number should be noted that biologically active of species. transcortin appeared earlier than that of Goodman and Moore (1971) delineated a TBG in the evolutional history of vertebrates cladogenetic tree of primate species based (Burlingame et al., 1965 ; Tanabe et al., on the analysis of their plasma proteins in 1970). an immunodiffusion system. The results of the present study on the immunoreactivity of primate transcortin and TBG are in very good accord with their cladgenetic tree (Fig. 4). From the cladogenetic tree, the Endocrinol. Japon. 150 SUEDA et al. April 1986

5636-5643. Acknowledgements Pugeat, M. M., G. P. Chrousos, B. C. Nisula, D. L. Loriaux, D. Brandon and M. B. Lipsett The authors wish to thank Dr. M. Minezawa (1984). Plasma cortisol transport and primate evolution. Endocrinology 115, 357-361. (The Primate Institute of Kyoto University), who Raymore, V. L. and R. W. Kuhn (1983). Homo- generously supplied plasma samples of non-human logous radioimmunoassay for rat cortisol- primate species to us. binding globulin. Endocrinology 112, 1091- This work was supported in part by a Grant- in-Aid for Scientific Research (No. 58570997) 1097. from the Japanese Ministry of Education, Sci- Robinson, P.A., C. Howkey and G.L. Hammond ence and Culture. (1985). A phylogenetic study of the structural and functional characteristics of corticosteroid binding globulin in primates. J. Endocri. 104, 251-257. References Seal, U. S. and R. P. Doe (1963). Corticosteroid- binding globulin: species distribution and Burlingame, A. L., P. Haug, T. Berdky and M. small-scale purification. Endocrinology 73, 371- Calvin (1965). Occurrence of biogenic steranes 376. and pentacyclic triterpanes in an eocene (52 Sueda, K., K. Ogawa and N. Matsui (1983). million years) and in an early precambrian Radioimmunoassay of human transcortin. shale (2.7 billion years): a preliminary report. Endocrinol. Japon. 30, 737-745. Proc. Natl. Acad. Sci. USA. 54, 1406-1412. Tanabe, Y., T. Ishii and Y. Tamaki (1970). Chrousos, G. P., D. Renquist, D. Brandon, C. Evolutionary aspects of plasma thyroxine-bind- Eil, M. Pugeat, R. Vigersky, G. B. Cutler, Jr., ing proteins of vertebrates. Gumma symposia D. L. Loriaux and M. B. Lipsett (1982). Gluco- on Endocrinology 7, 65-82. corticoid hormone resistance during primate Tanabe, Y., M. Ogawa and K. Nozawa (1974). evolution : Receptor-mediated Mechanisms. Polymorphism of thyroxine-binding prealbumin Proc. Natl. Acad. Sci. USA. 79, 2036-2040. (TBPA) in primate species. Japan. J. Genetics Goodman, M. and M. Moore (1971). Immuno- 49, 265-273. diffusion systematics of the primates. I. The Tanabe, Y. (1977). Transport proteins of thyro- Catarrhini. Systematic Zool. 20, 19-62. xine. in Biological science of hormone, Vol 2. Greenwood, F.C . and W. M. Hunter(1963). The Production and secretion of hormone, ed by preparation of 131I-labelled human growth Japan comparative endocrine society, Gakkai hormone of high specific radioactivity. Bio- shuppan senta publ., p. 172-173. chem. J., 89, 114-123. Yamamoto, S., S. Utsu, Y. Tanioka and N. Muldoon, T. G. and U. Westphal (1967). Steroid- Ohsawa (1977). Extremely high levels of corti- protein interactions. XV.Isolation and characte- costeroids and low levels of corticosteroid rization of corticosteroid-binding globulin binding macromolecule in plasma of marmoset from human plasma. J. Biol. Chem. 242, monkeys. Acta Endocrinol. 85, 398-405.