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Comparative studies on the amino acid composition of thyroglobulins from various lower and higher vertebrates: phylogenetic aspect
Comp. Biochem. Physiol., 1974, Vol. 49B,pp. 51 to 63. Pergamon Press. Printed in Great Britain
COMPARATIVE STUDIES ON THE AMINO ACID COMPOSITION OF THYROGLOBULINS FROM VARIOUS LOWER AND HIGHER VERTEBRATES: PHYLOGENETIC ASPECT A. BRISSON, J. M A R C H E L I D O N and F. L A C H I V E R Laboratoire de Physiologie g~n6rale et compar6e, Museum National d'Histoire Naturelle; et Laboratoire d'Endocrinologie compar6e associ6 au C.N.R.S., 7 rue Cuvier, 75005 Paris, France (Received 11 September 1973)
Al~tract--1. Thyroglobulins of five fishes, two reptiles and one mammal were purified by sucrose gradient and/or gel sepharose filtration. 2. Thyroglobulin amino acid analyses showed very significant variations in the content of numerous residues, especially Lys, Arg, Asp, Pro, Ala, IIe, Leu and Phe. 3. The results are discussed by considering the sedimentation coefficient values of the thyroglobulins investigated and the phylogenetic relationships of the species studied. INTRODUCTION RECENT ultracentrifugal investigations have indicated that the molecular organization of thyroid iodoproteins is similar through all classes of vertebrates. Thyroglobulin is the major iodoprotein of the thyroid gland from lower vertebrates to mammals (Lachiver et al., 1965, 1966; Salvatore et al., 1965; Roche et al., 1968). Previous studies from this laboratory have shown the existence of significant species differences between the sedimentation coefficients of fish thyroglobulins, which can account for 4-5 S (Lachiver et al., 1966). These interspecies differences were independent of the iodine content of the thyroglobulin (Brisson-Martin & Lachiver, 1970; Brisson, 1973). It could be suggested that these very important differences between sedimentation coefficients correspond to some variations in the primary and tertiary structures or also in the molecular weight of the thyroglobulin. To our knowledge, no report on amino acid analyses of non-mammalian thyroglobulins has been published, except the data of Hoshino & Ui (1970) on an amphibian and a chicken thyroglobulin and of Rolland et al. (1966) on a duck thyroglobulin. The purpose of this study was to determine the amino acid compositions of purified thyroglobulins from various fishes and reptiles belonging to different classes of lower vertebrates, in order to detect whether a relationship exists between 51
A. BRISSON, J. MARCHELIDON AND F. LACHIVER
52
the amino acid content of the thyroglobulin and the sedimentation coefficients of this protein. On the other hand, from a comparative point of view, it was of interest to investigate whether the amino acid compositions of the thyroglobulins studied reflect the phylogenetic relationships of various animal species in the evolutionary tree. Preliminary results of this work have been reported (Marchelidon et al., 1972). MATERIALS AND METHODS 1. Thyroid glands Thyroids or thyroid nodules from seven lower vertebrates and one mammal were used. The taxonomic list of the species investigated, sources and number of animals are given in Table 1. TABLE 1--TAXONOMICLIST AND SOURCEOF THE ANIMALSUSED Class Chondrichthyes Subclass Elasmobranehii Osteiehthyes Subclass Dipnoi Subclass Actinopterygii Superior order Teleostei Family Salmonides Family Anguillides
Taxonomic name Common name
Source
Scyliorhinus canicula L.
Dogfish
English Channel
Protopterus annectens L.
Lungfish
Tchad Lake
Salmo gairdnerii L. Salrno salar L. Anguilla anguilla L.
Trout
Pisciculture Gratereau Adour Somme
Reptilia Subclass Diapsida Superior order Lepidosauria Python s e b a e Superior order Archosauria
Mammalia Order Artiodactyla
Salmon Eel
Python
Crocodilus niloticus
Crocodile
Bos taurus
Ox
Zoological specimen, museum Zoological specimen, museum Slaughter-house
2. Preparation of the thyroid extracts Thyroid glands of each species were removed after exsanguination of the animals and pooled. Glands were quickly chilled, freed from extraneous tissue and were then frozen as necessary. Ox thyroids were sliced, then extracted overnight with 0"9 % NaCI (3 ml/g of tissue). The extract was clarified by centrifugation for 15 min at 30,000 g. Fish or reptile thyroids were homogenized in a Potter homogenizer with 0"I M NaCI-0-02 M sodium phosphate buffer, pH 7'4 (referred to as "NaCl phosphate buffer").
A M I N O A C I D C O M P O S I T I O N O F T H Y R O G L O B U L I N S F R O M VERTEBRATES
53
In several cases, glands labelled in vivo or in vitro with x~5I or lslI were used. T h e extracts or homogenates were centrifuged at 100,000 g for 1 hr in order to remove subcellular particles. All procedures were done at 4°C. 3. Isolation of thyroglobulin T h e supernatant solutions were purified by density gradient centrifugation as described by Salvatore et al. (1964) or by gel Sepharose 6 B filtration. T h e linear sucrose gradients were prepared at 4°C in NaCI phosphate buffer. T h e preparation was centrifuged at 25,000 rev/min for 23 hr (Rotor SW 25-1). T h e gel Sepharose 6 B filtration was chosen when larger amounts of protein (100-200 nag) were available. After spreading the sample on the top of the column, elution was carried out with NaC1 phosphate buffer at 4°C and at a flow rate adjusted to about 2 ml/cm 2 per hr. Fractions were collected in an automatic fraction collector (LKB). In these two methods, when the material studied was labelled, thyroglobulin was identified by measuring the radioactivity of each fraction collected. Otherwise, protein determinations were performed by measuring the absorbance at 280 nm. T h e appropriate fractions were pooled and then concentrated by vacuum filtration through a Visking dialysis bag at 4°C. Thyroglobulin solutions were then subjected to salting out between 1"4 and 2"2 M ammonium sulfate by the method of Derrien et al. (1948). A much higher concentration (2"7 M) was necessary to precipitate lungfish thyroglobulin. Finally, the thyroglobulin preparations were exhaustively dialyzed against 0-001 M NaCI solution and then lyophilized. Analyses of these purified thyroglobulins were performed by density gradient eentrifugation according to Martin & Ames (1961). Centrifugation was carried out in a Spinco Model La 65 B (41,000 rev/min for 7 hr) ultracentrifuge. In each fraction, after measurement of radioactivity, protein determination according to Lowry et al. (1951) and iodine assay were performed using a Technicon autoanalyzer. Sedimentation coefficients of purified thyroglobulins were calculated as described by Lachiver et al. (1966). Ox thyroglobulin in vitro labelled with t25I or tslI (sp. act. 10,000 counts/rain per/zg protein approximately) served as an internal standard, to which the value 19 S was assigned. In each case, the major protein peak had the same sedimentation coefficient and iodine content as those of the thyroglobulin identified in the crude extract. The slower sedimenting proteins (about 3-8 S) were always eliminated. 4. Amino acid determinations Approximately 250-1000/zg of each preparation were hydrolyzed in 6 N HCI in sealed tubes, under nitrogen, at l l 0 ° C , for 22 and 48 hr. since no further increase in most of the amino acids was observed after hydrolysis for a longer period of time (72 hr). T h e hydrolysates were analyzed according to Moore et al. (1958) using a Beckman autoanalyzer (Model 120 B). T h e water content of all thyroglobulins was evaluated as the weight loss of lyophilized ox thyroglobulin dried to a constant weight at 80°C in an oven. T h e value was found to account for 5 per cent. RESULTS
1. A m i n o acid compositions A m i n o acid d e t e r m i n a t i o n s w e r e p e r f o r m e d on several p u r i f i e d p r e p a r a t i o n s o f t h y r o g l o b u l i n o b t a i n e d f r o m each a n i m a l species. W h a t e v e r t h e p u r i f i c a t i o n m e t h o d used, t h e r e s u l t s w e r e q u i t e similar. A m i n o a c i d c o m p o s i t i o n s o f different t h y r o g l o b u l i n s s t u d i e d are g i v e n in T a b l e 2.
3.22 2"32 6"91 7.02 4-42 6"64 13"32 6"10 3"67 4"68 1"95 6"51 1"41 1 "90 9'40 2"54 4"57
86"58 0"724
Lys His Arg Asp T h r (*) Ser (*) Glu Pro Gly Ala ½Cys Val Met Ile Leu T yr Phe
Percentage recovery (t)
84"14 0"725
3.25 2"23 6.44 7"00 4"62 6"64 13"19 5"18 3"44 4"14 2"91 5"82 1"66 1 "88 8"80 2"58 4"36
Salmon
82"03 0"729
3.35 1 "81 7"05 7"69 4"59 5"77 12"42 5"30 3"37 4"30 1-47 5"65 1"29 2" 16 8'43 2"22 5"16
Eel
82"91 0"730
4.51 2" 19 5.44 8"34 4"75 5"63 11"84 4"78 3"41 3"24 1"66 5-13 1"24 3"65 8"02 3"08 6"00
Dogfish
83"86 0"726
4-78 2"22 4"21 8"59 5"20 7"37 12"03 4"22 2"99 2"88 2"70 5"53 1"38 3"43 7"26 2"96 6"11
Lungfish
84"56 0'750
4.61 1 "20 5.31 8.50 5'56 6-01 12"72 4"60 3"53 3"20 3"73 4"20 1"10 3"57 7-75 2"94 6"03
Python
70-22 0"760
3.95 1 "22 4"83 7"70 3-60 3"06 11"04 4"43 2"70 2"71 2"50 4"05 1"04 3"12 6"76 2-31 5"20
Crocodile
OBTAINED FROM VARIOUS ANIMAL SPECIES
Species
OF PURIFIED THYROGLOBULINS
85'99 0"724
2.79 1"51 8.04 6.25 4'09 6"94 13"23 6-16 3-42 4"87 3'35 5'14 0'97 2' 19 8"33 2'77 5'94
Ox
The tryptophan percentage was assumed to be 3 per cent.
~wl~w.
Values are expressed as rag/100 mg of dry protein hydrolyzed. Analyses were carried out at least in duplicate for each hydrolysis time. Values represent the average of two determinations. * Values were extrapolated at zero time by the method of least squares, in order to correct for hydrolytic losses. t T he partial specific volumes of the thyroglobulins 17 were evaluated according to Cohn & Edsall (1943), from the partial specific volume ~ and dry weight per cent protein of each amino acid w in the thyroglobulin, by the equation: 17 =
Trout
ACID COMPOSITION
Amino acids
TABLE 2--AMINO
t~
O
>~
.~
Z
~>
Z
-~
~"
-~
A M I N O A C I D C O M P O S I T I O N O F T H Y R O G L O B U L I N S F R O M VERTEBRATES
55
The values represent the averages of the maximum amounts obtained for each amino acid, after hydrolysis for 22 or 48 hr. They are expressed as rag/100 mg of dry protein. Iodoamino acids, tryptophan and amids were not determined. Except in the case of crocodile thyroglobulin the percentage recovery after hydrolysis of various preparations was about 84 per cent of the dry weight of the protein hydrolyzed. The partial specific volumes of various thyroglobulins evaluated according to Cohn and Edsall (1943) were little different from one another. It must be noted that the data obtained for the amino acid composition of ox thyroglobulin were in good agreement with those reported by other authors (Piez & Morris, 1960; Rolland et al., 1966; Spiro, 1970). On the whole, the amino acid compositions of the different lower vertebrate thyroglobulins investigated indicated some degree of similarity between each one and ox thyroglobulin composition. However, a statistical study of the results presented in Table 2 displayed significant differences in the contents of numerous amino acids of the various thyroglobulins studied (Table 3). Comparisons of the amino acid compositions of these thyroglobulins have been made by considering the phylogenetic relationships of species, on the one hand, and the values of sedimentation coefficients of the thyroglobulins, on the other. Variance analysis was carried out for each amino acid, according to F-test of Fischer. From Table 3, it may be seen that: The salmonid (trout and salmon) thyroglobulins whose sedimentation coefficient is 17.7 S have the closest amino acid compositions. The comparison of eel thyroglobulin (19-2 S) with that of salmonides (17.7 S) (these three fish species belonging to the Teleostei superior order) shows only a very highly significant variation for histidine. The thyroglobulins of lungfish and salmonides, representative of Dipnoi and Actinopterygii subclasses respectively, display the extreme values of sedimentation coefficient (22-5 and 17.7 S) and show very different amino acid compositions. The variations for seven residues are very highly significant. The comparisons between thyroglobulins of different fish classes (dogfish v. salmonides or dogfish v. lungfish) would suggest that the more different the sedimentation coefficients, the higher the number of significant variations between thyroglobulins investigated. The same would be true if the amino acid composition of ox thyroglobulin was compared with that of the different fish thyroglobulins. The thyroglobulins of the two reptiles studied, crocodile and python, with sedimentation coefficients of about 19 S, present amino acid compositions relatively close to each other. But, in contrast to the above and therefore independently of sedimentation coefficient values, the amino acid composition of reptile thyroglobulins shows a great similarity to that of dogfish and lungfish thyroglobulins,
---
Tyr Phe
.
.
-S
~ S
--
~ S
S -----
--
-VHS -.
.
.
-VHS
VHS
VHS
~ S
-VHS S S VHS
VHS
S . VHS
Lungfish 22-5 S Salmonides 17"7 S
.
.
. S
S VHS
VHS
VHS
VHS
~ S VHS --VHS
.
VHS
S
.
.
Dogfish 21 S Salmonides 17"7 S
---
HS
. HS
--
S --HS ~ S
--
-. VHS
Dogfish 21 S Lungfish 22"5 S
.
-HS
--
--
--
HS S ----
HS H S
S
--
Crocodile 18"6 S Python 19"5 S
HS, highly significant, 1
S, s i g n i f i c a n t , c l o s e t o P = 5 p e r c e n t . S, s i g n i f i c a n t , 5 < P < 1 per cent.
sedimentation coefficients of various thryoglobulins studied. values obtained for ½Cys were not near enough to allow for a statistical calculation.
S
Leu
* The ]" T h e
. --
Met Iso
--S ---
Ser Glu Pro Gly Ala
S
-.
Asp Thr
½Cys'~ Val
--S
acids
Lys His Arg
Amino
Eel 19"2 S Salmonides 17"7 S
Species
.
-HS
VHS
VHS
VHS
VHS HS HS HS VHS
S VH S
S --
--
--
VHS
S -----
-H S
S VHS --
Lungfish+ Dogfish
Salmonides VHS VHS VHS
Reptiles
Reptiles
ANALYSIS OF A M I N O ACID C O M P O S I T I O N S OF VARIOUS L O W E R VERTEBRATE T H Y R O G L O B U L I N S S T U D I E D
Trout 17"7 S * Salmon 17-7 S
TABLE 3--VARIANCE
t~ ~0
z
z
©
O
z
>
5"4
7"5
5"4 9-4 12.8 7.8
Arg
Asp
Thr Ser Glu Pro
9-8 1"7 4"6
1"3 2-5
1-8 7-5
7.7 7"9
5"9 8.7 12.7 7"2
8"8
5"9
1"7
3"4
Eel
9-3 2"4 5"3
1-2 4"2
2"1 6"8
7.8 6.0
6"2 8.5 12.1 6-5
9"5
4"5
2"1
4"6
Dogfish
8-3 2-3 5"4
1-3 3"9
3"4 7"2
6.8 5-2
6"7 11.0 12.1 5.6
9-7
3"5
2"1
4"8
Lungfish
9"3 2-5 5-9
1"8 4"2
2"4 6"2
7.3 5"7
5"7 9.7 11.7 5.7
10"5
4"0
2"3
5"0
Xenope
8-9 2"3 5"3
1"1 4-0
4"0 5"5
7"9 5.8
7"0 9-0 12-8 6.1
9-7
4"4
1"1
4"7
Python
9"5 2"3 5"6
1"3 4"4
3-1 6"4
7"6 6-1
5"8 5.0 13-7 7.2
11-0
5"0
1"4
4-9
Crocodile
9"2 3"4 5-3
1"5 3"8
4"0 6"1
7"9 6.1
5"2 10-1 13"5 5-3
9"2
4"7
1"1
3-9
Duck*
8"9 3"2 5-5
1"3 4"2
2"6 5-8
7"3 6"3
5"3 8.9 12.2 9.7
9"6
4"2
1"1
3"7
Chickent
9"2 2"1 5"0
0"9 2"4
4"1 6"5
7.5 8-6
5"0 10.0 12"8 7"9
6-8
6"4
1"3
2"7
Ox
Results are expressed as moles/100 moles of total amino acids. t Values reported by Hoshino & Ui (1970). * Values reported by Rolland et al. (1966). Mean of values obtained by Pierce et al. (1965), Rolland et al. (1966), Hoshino & Ui (1970) and Spiro (1970).
9"9 2-0 3"7
10-3 1"9 3"8
Tyr Phe
Leu
1"6 2"1
1-3 2"0
Met Ile
3"6 7.5
2-3 8"1
7.7 7"4
5"8 9.7 13.1 6.8
7"7
5"2
2-0
3'2
Salmon
Species
A M I N O ACID COMPOSITIONS OF PURIFIED THYROGLOBULINS OBTAINED FROM VARIOUS ~ R T E B R A T E CLASSES
~Cys Val
Ala
7"9 8.1
2"0
His
Gly
3"1
Lys
Amino acids T r o u t
TABLE 4
9-2 2"1 5"0
1"3 2"9
3-8 6-1
7"6 7-4
5"3 9-0 13-3 6-5
7"6
5"4
1"4
3"5
Humans
L/I ".-.I
0
Z o
8
0
>
O
58
A. BRISSON,J. MARCHELIDONANDF. LACHIVER
whereas it is very differentfrom salmonidthyroglobulins, since the contents of all amino acids except tyrosine differ significantly.
2. Comparison of the amino acid composition of thyroglobulinsfrom different vertebrate classes The results obtained for various thyroglobulins studied and listed in Table 4 are expressed as moles/100 moles of total amino acid recovered, in order to compare them with those given by other authors (Rolland et al., 1966; Hoshino & Ui, 1970) for the thyroglobulins of one amphibian (Xenopus laevis), two birds (Gallus gallus; Anas platyrhynchos) and one mammal (Homo sapiens). It is seen that a noteworthy degree of similarity exists between the thyroglobulins of xenope, python, crocodile, chicken, duck and those of dogfish and lungfish. The amino acid compositions of all these vertebrate thyroglobulins differ widely from those of teleost and mammalian thyroglobulins, especially for the content of Lys, Arg, Asp, Pro and Ile. The teleost thyroglobulins are relatively homogeneous. Their amino acid compositions are closer to mammalian thyroglobulins, from which they differ chiefly for the valine and phenylalanine contents than that of the other fish thyroglobulins studied (lungfish and dogfish). Considering all the thyroglobulins together, approximately 50 per cent of the total residues are aliphatic amino acids, while the basic amino acids account for 10-11 per cent and the dicarboxylic amino acids make up 20 per cent of amino acid residues. It is of interest that, although the contents of each basic amino acid vary widely according to the species studied, the total amount of basic residues differs little. DISCUSSION Several immunological studies, especially on mammalian thyroglobulins, have provided evidence about the species specificity of thyroglobulin (Hecktoen et al., 1927; Adant & Spehl, 1934; Stokinger & Heidelberger, 1937; Yagi & Kodama, 1955; Martinelli et al., 1969; Hoshino & Ui, 1970). The quantitative precipitin analysis and double-diffusion tests have. indicated that mammalian thyroglobulins are more or less related immunologically to each other, but no cross-reaction has been shown between mammalian thyroglobulins and those from two other vertebrate classes, Aves and Amphibia (Hoshino & Ui, 1970). For our part, the existence of significant differences between the sedimentation coefficients of fish thyroglobulins (Lachiver et aL, 1966) suggested possible species variations in the structure of these proteins and fitted in with the hypothesis of thyroglobulin specifieity. On the other hand, we have determined the amino acid composition of thyroglobulins from various lower vertebrate classes (Chondrichthyes, Osteichthyes, Reptilia).
AMINO ACID COMPOSITION OF THYROGLOBULINS FROM VERTEBRATES
59
The amino acid analyses of several mammalian thyroglobulins have been performed by different authors in recent years (Pierce, 1965; Rolland et al., 1966; Spiro, 1970; Hoshino & Ui, 1970), but hitherto the non-mammalian thyroglobulins have been the subject of very few studies: only the thyroglobulins from one amphibian and two birds have been investigated by Rolland et aL (1966) and Hoshino & Ui (1970). These proteins have been found to be rather different from those of mammals. In the present work, amino acid analyses of thyroglobulins from various fish and reptile species have indicated some similarity in the general composition of these purified proteins with thyroglobulins from other vertebrate classes. These results thus provide evidence of the homology (Florkin, 1962) of the major thyroidal protein in the whole zoological gnasthastome vertebrate series. However, these homologous proteins are not identical. Indeed a detailed examination of data obtained (Tables 2 and 3) has displayed the existence of significant interspecies variations in the contents of numerous amino acids and especially of basic (Lys, Arg), acid (Asp) and hydrophobic (Pro, Ala, Ile, Leu, Phe) residues. A variance analysis of preliminary results (Marchelidon et aL, 1972) has suggested that the more different the sedimentation coefficient thyroglobulins, the more distinct the amino acid composkions of these proteins. Yet, a comparison of data concerning the thyroglobulins from different vertebrate classes has shown that the importance of amino acid variations is independent of sedimentation coefficient differences. Indeed a great similarity of amino acid composition has been observed between the amphibian, reptile and bird thyroglobulins, having sedimentation coefficients close to 19 S, and the dogfish and lungfish thyroglobulins, whose sedimentation coefficients were definitely higher than 19 S (i.e., 21 and 22.5 S respectively). It is possible that, despite similar composkions, these various protein molecules could present variable amino acid sequences and therefore distinct conformational structures. Nevertheless, as in the case of other classes of homologous proteins, the ratios of hydrophobicity to fractional charge calculated, as reported by Welscher (1969), for the various thyroglobulins investigated, fall within very narrow limits, and the total number of hydrophobic amino acids is nearly constant. These data argue for close secondary and tertiary structures (Rolland et aL, 1966). On the assumption that these various proteins are not too different in shape and have partial specific volumes which are approximately identical, the hypothesis of molecular weight variations can be suggested. A crude estimate of the apparent molecular weight (mw) of various thyroglobulins can be obtained from the formula of Schachman (1959): mw2_/S~, mwl \ S J where mWx and S x are the molecular weight (670,000) and sedimentation coefficient (19 S) of ox thyroglobulin. For example, lungfish and salmonid thyroglobulins would have a molecular weight of 860,000 and 600,000 respectively.
60
A. BRISSON, J. MARCHELIDON AND F. LACHIVER
Would the number of polypeptide subunits of this protein change in some species ? But as long as the number and size of thyroglobulin subunits are a controversial subject (see Bornet, 1971; Rolland & Lissitzky, 1972) no serious interpretation of our data can be given. On the other hand, in considering the evolutionary tree of vertebrates (Fig. 1), thyroglobulin amino acid compositions seem to be connected with phylogenetic relationships between the various animal species investigated.
/ (Pyth0n,Croc0dite)
HOLO$TEI
/
CROSSOPTERY611 / >OlPflOI (Lungfish)
CHONDR(]~
ACTINOPTERYGil , CHOAHICHTYES \ v /
"
/
/
CHOHpRIGHTHYE$ ~ (Dogfish)
Fig:1 A simplified pbylogenetictree of the gnathostomevertebrates outlined from ROMER(1949) and GRASSE(1958) * 1he namesof vertebrate classesare underlined It can be seen that the composition of dogfish and lungfish thyroglobulins are closer to amphibian and reptile thyroglobulins than to that of other fishes such as the teleosts, but the forms arranged in the Pisces superclass belong in fact to three independent evolutive lines: the Chondrichthyes, the Actinopterygii and the Choaniehthyes, the two end-branches of which are represented by the classes of Aves and Mammalia. Table 4 shows that the various thyroglobulins studied can be classified into three groups: teleost, mammals and other vertebrates. From a phylogenetic point of view, the evolution of the structure of different proteins having a lower molecular weight (such as hemoglobin, cytochrome, insulin) has been studied (see Acher, 1957; Neuman & Humbel, 1969; Jukes, 1971; Diekerson, 1971). Substitution and duplication events would be essential in protein evolution and could breed several evolutive lines from a common ancestral molecule. From these data, it may be supposed that thyroglobulin composition would have varied independently in the Actinopterygii and Mammalia branches of
AMINO ACID COMPOSITION OF THYROGLOBULINS FROM VERTEBRATES
61
the evolutionary tree, whereas it has remained closer to the composition of the ancestral molecule in the Chondrichthyes, Choanichthyes, Amphibia, Reptilia and Aves. SUMMARY 1. Thyroglobulins of five fishes (trout, salmon, eel, dogfish, lungfish), two reptiles (python, crocodile) and one mammal (ox) were purified. 2. The amino acid composition of these purified thyroglobulins has been carried out. A comparison of results obtained with thyroglobulins from other vertebrate classes has shown the homology of the thyroglobulin throughout the series of vertebrates, from lower vertebrates to mammals. 3. However, a variance analysis has pointed out very significant variations of the numerous amino acid contents (especially Arg, Lys, Asp, Pro, Ala, Ile, Leu and Phe). 4. The importance of amino acid composition variations is independent of sedimentation coefficient differences of the thyroglobulins studied. 5. On the contrary, the variations of amino acid compositions seem to be correlated to the phylogenetic relationships of the species investigated. Acknowledgements--The authors wish to express their thanks to Miss Boulu Franqoise for her valuable technical assistance.
REFERENCES
ACI-IERR. (1967) Evolution de la structure des prot~ines. Bull. Soc. chim. Biol. 49, 609-631. ADXNTM. & SPEHLP. (1934) La sp~cificit6 immunologique des thyroglobulines. C. R. Soc. Biol. 117, 230-231. BORNm" H. (1971) Nouvelles m&hodes d'~tude de la thyroglobuline en milieu dodecyl sulfate. Colloque: M&hodologie exp6rimentale en physiologic et en physiopathologie thyro'idiennes, pp. 169-183. (Edited by MORN~ R. & N u ~ z J.), Lyon. BRISSON-MARTIN A. & LACHIVERF. (1970) Variations sp6cifiques du coefficient de s6dimentation de la pr&hyroglobuline chez quelques Vert6br~s inf6rieurs. C. R. Soc. Biol. 164, 1922-1925. BRISSONA. (1973) Etude compar6e de la biosynth6se et de l'iodation des prot~ines thyro~diennes chez quelques Vert6br6s inf6rieurs. Sur l'existence de differences sp~cifiques des thyroglobulines. Th~se de Sciences, Paris. No. enregistrement C.N.R.S.A.O. 7963. COliN E. J. & EDS~L J. T. (1943) Density and apparent specific volume of proteins. In Proteins, Amino Acids andPeptides (Edited by Com'~ E. J. & EDSALLJ. T.), pp. 370-381. Reinhold, New York. DFmRI~ Y., MICHELR. & ROCHEJ. (1948) Recherches sur la pr6paration et les propri&~s de la thyroglobuline pure. Biochim. biophys. Acta 2, 454-470. DICKERSONR. E. (1971) The structure of cytochrome c and the rates of molecular evolution. J. molec. Evolution 1, 26-45. FLORKINM. (1962) Isologie, homologie, analogie et convergence en biochimie compar~e. Bull. Classe Sci. Acad. Roy. Belg. 48, 819-824. GRASS~P. P. (1958) Classification des Vertdbrds: Traitd de Zoologie, 8th edn., 1st fast., pp. 1-10. Masson et Cie, Paris.
62
A. BRISSON, J. MARCHELIDONAND F. LACHIVER
HEKTOEN L., F o x H. & SCHULHOFK. (1927) Specificness in the precipitin reaction..7. Infect. Dis. 40, 641-646. HOSHINO W. & UI N. (1970) Comparative studies on the properties of thyroglobulin from various animal species. Endocrinologiajaponica 17, 521-533. JUKES T. H. (1971) Comparisons of the polypeptide chains of globins..7, molec. Evolution 1, 46-62. LACHIVER F., FONTAINEY. A. & MARTIN A. (1965) The iodination in vivo of thyroid proteins in various Vertebrates. In Current Topics in Thyroid Research (Edited by CASSANOC. & ANDm~OLI M.), pp. 182-192. Academic Press, New York. LACmWR F., MARTIN A. & FONTAINEM. (1966) Iodation in vivo des prot6ines thyroidiennes chez quelques Vertdbr6s inf6rieurs. Gunma Symposia on Endocrinology 3, 109-120. LOWRY O. H., ROSEBROUCHN. J., FARR A. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent, dY. biol. Chem. 193, 265-275. MARCHELIDON J., BRISSON-MARTINA. & LACHIVER F. (1972) Composition en acides amines de la thyroglobuline de divers Poissons. Congr6s Europ. d'Endocrinologie compar6e. 6~me Montpellier 1971. Gen. & compar. Endocr. 18, 601. MARTIN R. G. & AMES B. M. (1961) A method for determining the sedimentation behavior of enzymes: application to protein mixtures..7, biol. Chem. 236, 1372-1379. MARTINELLI G., PENTIMALLID., PERRONE N. & ZAPPACOSTAS. (1969) Studi sulla specificith di specie della tireoglobulina. Atti Soc. Ital. Patol. Communicazione tenuta all' X I Congresso della societ~ italiana di patologia, Roma. MOORE S., SPACKMAND. H. & STEIN W. H. (1958) Chromatography of amino acids on sulfonated polystyrene resins. An improved system. Analyt. Chem. 30, 1185-1190. NEUMAN P. A. & HUMBEL R. E. (1969) Isolation of a single component of fish insulin from a bonito-tuna-swordfish insuline mixture and its complete amino acid sequence. Int..7. Prot. Res. 1, 125-140. PIERCE J. G., RAWlTCH A. B., BROWN D. M. & STANLEYP. G. (1965) Human thyroglobulin: studies on the native and 5-carboxymethylated protein. Biochim. biophys. Acta 111, 247-257. PIEZ K. A. & MORRIS L. (1960) Modified procedure for the automatic analysis of amino acids. Analyt. Biochem. 1, 187-201. ROCHE J., SALVATOREG., SENA L., ALOJ J. & COVELLI I. (1968) Thyroid iodoproteins in Vertebrates: ultracentrifugal pattern and iodination rate. Comp. Biochem. Physiol. 27, 67-82. ROLLAND M., BISMUTH J., FONDARIJ. & LISSITZKYS. (1966) Composition en acides amin6s de la thyroglobuline de diff6rentes esp~ces animales. Acta Endocr. 53, 286-296. ROLLAND M. & LlSSlTZKY S. (1972) Polypeptides non covalently associated in 19 S thyroglobulin. Biochim. biophys. Acta 278, 316-336. ROMER A. S. (1949) The Vertebrate Body, pp. 30-64. W. B. Saunders, Philadelphia. SALVATOREG., SALVATOREM., CAHMANNH. J. & ROBBINS J. (1964) Separation thyroidal iodoproteins and purification of thyroglobulin by gel filtration and density gradient centrifugation, o7. biol. Chem. 239, 3267-3274. SALVATOREG., SENA L., VlSClDI E. & SALVATOREM. (1965) T h e thyroid iodoproteins 12 S, 19 S and 27 S in various animal species and their physiological significance. In Current Topics in Thyroid Research (Edited by CASSANO C. & ANDREOLI M.), pp. 193-206. Academic Press, New York. SCHACHMAN H. K. (1959) Ultracentrifugation in Biochemistry. Academic Press, New York. SPIRO M. J. (1970) Studies on the protein portion of thyroglobulin. Amino acid compositions and terminal amino acids of several thyroglobulins..7, biol. Chem. 245, 2820-2826. STOKINGER H. E. & HEIDELBERGER M. (1937) A quantitative theory of the precipitin react i o n - - V I . T h e reaction between mammalian thyroglobulins and antibodies to homologous and heterologous preparations. J. exp. Med. 66, 251-272.
A M I N O ACID C O M P O S I T I O N OF THYROGLOBULINS FROM VERTEBRATES
63
WE~Cm~R H. D. (1969) Correlations between amino acid sequence and conformation of immunoglobulin light chains--I. Hydrophobicity and fractional charge. Int. J. Prot. Res. 1, 253-265. YAGI Y. 8z KODAMA M. (1955) Etudes immunochimiques des prot6ines thyroidiennes. C. R. Soc. Biol. 149, 2282-2284. Key Word Index--Thyroglobulin; amino acid composition; lower vertebrates; phylogenetic relationships of thyroglobulins; thyroglobulin specificity; sedimentation coefficient thyroglobulin.
COMPARATIVE STUDIES ON THE AMINO ACID COMPOSITION OF THYROGLOBULINS FROM VARIOUS LOWER AND HIGHER VERTEBRATES: PHYLOGENETIC ASPECT A. BRISSON, J. M A R C H E L I D O N and F. L A C H I V E R Laboratoire de Physiologie g~n6rale et compar6e, Museum National d'Histoire Naturelle; et Laboratoire d'Endocrinologie compar6e associ6 au C.N.R.S., 7 rue Cuvier, 75005 Paris, France (Received 11 September 1973)
Al~tract--1. Thyroglobulins of five fishes, two reptiles and one mammal were purified by sucrose gradient and/or gel sepharose filtration. 2. Thyroglobulin amino acid analyses showed very significant variations in the content of numerous residues, especially Lys, Arg, Asp, Pro, Ala, IIe, Leu and Phe. 3. The results are discussed by considering the sedimentation coefficient values of the thyroglobulins investigated and the phylogenetic relationships of the species studied. INTRODUCTION RECENT ultracentrifugal investigations have indicated that the molecular organization of thyroid iodoproteins is similar through all classes of vertebrates. Thyroglobulin is the major iodoprotein of the thyroid gland from lower vertebrates to mammals (Lachiver et al., 1965, 1966; Salvatore et al., 1965; Roche et al., 1968). Previous studies from this laboratory have shown the existence of significant species differences between the sedimentation coefficients of fish thyroglobulins, which can account for 4-5 S (Lachiver et al., 1966). These interspecies differences were independent of the iodine content of the thyroglobulin (Brisson-Martin & Lachiver, 1970; Brisson, 1973). It could be suggested that these very important differences between sedimentation coefficients correspond to some variations in the primary and tertiary structures or also in the molecular weight of the thyroglobulin. To our knowledge, no report on amino acid analyses of non-mammalian thyroglobulins has been published, except the data of Hoshino & Ui (1970) on an amphibian and a chicken thyroglobulin and of Rolland et al. (1966) on a duck thyroglobulin. The purpose of this study was to determine the amino acid compositions of purified thyroglobulins from various fishes and reptiles belonging to different classes of lower vertebrates, in order to detect whether a relationship exists between 51
A. BRISSON, J. MARCHELIDON AND F. LACHIVER
52
the amino acid content of the thyroglobulin and the sedimentation coefficients of this protein. On the other hand, from a comparative point of view, it was of interest to investigate whether the amino acid compositions of the thyroglobulins studied reflect the phylogenetic relationships of various animal species in the evolutionary tree. Preliminary results of this work have been reported (Marchelidon et al., 1972). MATERIALS AND METHODS 1. Thyroid glands Thyroids or thyroid nodules from seven lower vertebrates and one mammal were used. The taxonomic list of the species investigated, sources and number of animals are given in Table 1. TABLE 1--TAXONOMICLIST AND SOURCEOF THE ANIMALSUSED Class Chondrichthyes Subclass Elasmobranehii Osteiehthyes Subclass Dipnoi Subclass Actinopterygii Superior order Teleostei Family Salmonides Family Anguillides
Taxonomic name Common name
Source
Scyliorhinus canicula L.
Dogfish
English Channel
Protopterus annectens L.
Lungfish
Tchad Lake
Salmo gairdnerii L. Salrno salar L. Anguilla anguilla L.
Trout
Pisciculture Gratereau Adour Somme
Reptilia Subclass Diapsida Superior order Lepidosauria Python s e b a e Superior order Archosauria
Mammalia Order Artiodactyla
Salmon Eel
Python
Crocodilus niloticus
Crocodile
Bos taurus
Ox
Zoological specimen, museum Zoological specimen, museum Slaughter-house
2. Preparation of the thyroid extracts Thyroid glands of each species were removed after exsanguination of the animals and pooled. Glands were quickly chilled, freed from extraneous tissue and were then frozen as necessary. Ox thyroids were sliced, then extracted overnight with 0"9 % NaCI (3 ml/g of tissue). The extract was clarified by centrifugation for 15 min at 30,000 g. Fish or reptile thyroids were homogenized in a Potter homogenizer with 0"I M NaCI-0-02 M sodium phosphate buffer, pH 7'4 (referred to as "NaCl phosphate buffer").
A M I N O A C I D C O M P O S I T I O N O F T H Y R O G L O B U L I N S F R O M VERTEBRATES
53
In several cases, glands labelled in vivo or in vitro with x~5I or lslI were used. T h e extracts or homogenates were centrifuged at 100,000 g for 1 hr in order to remove subcellular particles. All procedures were done at 4°C. 3. Isolation of thyroglobulin T h e supernatant solutions were purified by density gradient centrifugation as described by Salvatore et al. (1964) or by gel Sepharose 6 B filtration. T h e linear sucrose gradients were prepared at 4°C in NaCI phosphate buffer. T h e preparation was centrifuged at 25,000 rev/min for 23 hr (Rotor SW 25-1). T h e gel Sepharose 6 B filtration was chosen when larger amounts of protein (100-200 nag) were available. After spreading the sample on the top of the column, elution was carried out with NaC1 phosphate buffer at 4°C and at a flow rate adjusted to about 2 ml/cm 2 per hr. Fractions were collected in an automatic fraction collector (LKB). In these two methods, when the material studied was labelled, thyroglobulin was identified by measuring the radioactivity of each fraction collected. Otherwise, protein determinations were performed by measuring the absorbance at 280 nm. T h e appropriate fractions were pooled and then concentrated by vacuum filtration through a Visking dialysis bag at 4°C. Thyroglobulin solutions were then subjected to salting out between 1"4 and 2"2 M ammonium sulfate by the method of Derrien et al. (1948). A much higher concentration (2"7 M) was necessary to precipitate lungfish thyroglobulin. Finally, the thyroglobulin preparations were exhaustively dialyzed against 0-001 M NaCI solution and then lyophilized. Analyses of these purified thyroglobulins were performed by density gradient eentrifugation according to Martin & Ames (1961). Centrifugation was carried out in a Spinco Model La 65 B (41,000 rev/min for 7 hr) ultracentrifuge. In each fraction, after measurement of radioactivity, protein determination according to Lowry et al. (1951) and iodine assay were performed using a Technicon autoanalyzer. Sedimentation coefficients of purified thyroglobulins were calculated as described by Lachiver et al. (1966). Ox thyroglobulin in vitro labelled with t25I or tslI (sp. act. 10,000 counts/rain per/zg protein approximately) served as an internal standard, to which the value 19 S was assigned. In each case, the major protein peak had the same sedimentation coefficient and iodine content as those of the thyroglobulin identified in the crude extract. The slower sedimenting proteins (about 3-8 S) were always eliminated. 4. Amino acid determinations Approximately 250-1000/zg of each preparation were hydrolyzed in 6 N HCI in sealed tubes, under nitrogen, at l l 0 ° C , for 22 and 48 hr. since no further increase in most of the amino acids was observed after hydrolysis for a longer period of time (72 hr). T h e hydrolysates were analyzed according to Moore et al. (1958) using a Beckman autoanalyzer (Model 120 B). T h e water content of all thyroglobulins was evaluated as the weight loss of lyophilized ox thyroglobulin dried to a constant weight at 80°C in an oven. T h e value was found to account for 5 per cent. RESULTS
1. A m i n o acid compositions A m i n o acid d e t e r m i n a t i o n s w e r e p e r f o r m e d on several p u r i f i e d p r e p a r a t i o n s o f t h y r o g l o b u l i n o b t a i n e d f r o m each a n i m a l species. W h a t e v e r t h e p u r i f i c a t i o n m e t h o d used, t h e r e s u l t s w e r e q u i t e similar. A m i n o a c i d c o m p o s i t i o n s o f different t h y r o g l o b u l i n s s t u d i e d are g i v e n in T a b l e 2.
3.22 2"32 6"91 7.02 4-42 6"64 13"32 6"10 3"67 4"68 1"95 6"51 1"41 1 "90 9'40 2"54 4"57
86"58 0"724
Lys His Arg Asp T h r (*) Ser (*) Glu Pro Gly Ala ½Cys Val Met Ile Leu T yr Phe
Percentage recovery (t)
84"14 0"725
3.25 2"23 6.44 7"00 4"62 6"64 13"19 5"18 3"44 4"14 2"91 5"82 1"66 1 "88 8"80 2"58 4"36
Salmon
82"03 0"729
3.35 1 "81 7"05 7"69 4"59 5"77 12"42 5"30 3"37 4"30 1-47 5"65 1"29 2" 16 8'43 2"22 5"16
Eel
82"91 0"730
4.51 2" 19 5.44 8"34 4"75 5"63 11"84 4"78 3"41 3"24 1"66 5-13 1"24 3"65 8"02 3"08 6"00
Dogfish
83"86 0"726
4-78 2"22 4"21 8"59 5"20 7"37 12"03 4"22 2"99 2"88 2"70 5"53 1"38 3"43 7"26 2"96 6"11
Lungfish
84"56 0'750
4.61 1 "20 5.31 8.50 5'56 6-01 12"72 4"60 3"53 3"20 3"73 4"20 1"10 3"57 7-75 2"94 6"03
Python
70-22 0"760
3.95 1 "22 4"83 7"70 3-60 3"06 11"04 4"43 2"70 2"71 2"50 4"05 1"04 3"12 6"76 2-31 5"20
Crocodile
OBTAINED FROM VARIOUS ANIMAL SPECIES
Species
OF PURIFIED THYROGLOBULINS
85'99 0"724
2.79 1"51 8.04 6.25 4'09 6"94 13"23 6-16 3-42 4"87 3'35 5'14 0'97 2' 19 8"33 2'77 5'94
Ox
The tryptophan percentage was assumed to be 3 per cent.
~wl~w.
Values are expressed as rag/100 mg of dry protein hydrolyzed. Analyses were carried out at least in duplicate for each hydrolysis time. Values represent the average of two determinations. * Values were extrapolated at zero time by the method of least squares, in order to correct for hydrolytic losses. t T he partial specific volumes of the thyroglobulins 17 were evaluated according to Cohn & Edsall (1943), from the partial specific volume ~ and dry weight per cent protein of each amino acid w in the thyroglobulin, by the equation: 17 =
Trout
ACID COMPOSITION
Amino acids
TABLE 2--AMINO
t~
O
>~
.~
Z
~>
Z
-~
~"
-~
A M I N O A C I D C O M P O S I T I O N O F T H Y R O G L O B U L I N S F R O M VERTEBRATES
55
The values represent the averages of the maximum amounts obtained for each amino acid, after hydrolysis for 22 or 48 hr. They are expressed as rag/100 mg of dry protein. Iodoamino acids, tryptophan and amids were not determined. Except in the case of crocodile thyroglobulin the percentage recovery after hydrolysis of various preparations was about 84 per cent of the dry weight of the protein hydrolyzed. The partial specific volumes of various thyroglobulins evaluated according to Cohn and Edsall (1943) were little different from one another. It must be noted that the data obtained for the amino acid composition of ox thyroglobulin were in good agreement with those reported by other authors (Piez & Morris, 1960; Rolland et al., 1966; Spiro, 1970). On the whole, the amino acid compositions of the different lower vertebrate thyroglobulins investigated indicated some degree of similarity between each one and ox thyroglobulin composition. However, a statistical study of the results presented in Table 2 displayed significant differences in the contents of numerous amino acids of the various thyroglobulins studied (Table 3). Comparisons of the amino acid compositions of these thyroglobulins have been made by considering the phylogenetic relationships of species, on the one hand, and the values of sedimentation coefficients of the thyroglobulins, on the other. Variance analysis was carried out for each amino acid, according to F-test of Fischer. From Table 3, it may be seen that: The salmonid (trout and salmon) thyroglobulins whose sedimentation coefficient is 17.7 S have the closest amino acid compositions. The comparison of eel thyroglobulin (19-2 S) with that of salmonides (17.7 S) (these three fish species belonging to the Teleostei superior order) shows only a very highly significant variation for histidine. The thyroglobulins of lungfish and salmonides, representative of Dipnoi and Actinopterygii subclasses respectively, display the extreme values of sedimentation coefficient (22-5 and 17.7 S) and show very different amino acid compositions. The variations for seven residues are very highly significant. The comparisons between thyroglobulins of different fish classes (dogfish v. salmonides or dogfish v. lungfish) would suggest that the more different the sedimentation coefficients, the higher the number of significant variations between thyroglobulins investigated. The same would be true if the amino acid composition of ox thyroglobulin was compared with that of the different fish thyroglobulins. The thyroglobulins of the two reptiles studied, crocodile and python, with sedimentation coefficients of about 19 S, present amino acid compositions relatively close to each other. But, in contrast to the above and therefore independently of sedimentation coefficient values, the amino acid composition of reptile thyroglobulins shows a great similarity to that of dogfish and lungfish thyroglobulins,
---
Tyr Phe
.
.
-S
~ S
--
~ S
S -----
--
-VHS -.
.
.
-VHS
VHS
VHS
~ S
-VHS S S VHS
VHS
S . VHS
Lungfish 22-5 S Salmonides 17"7 S
.
.
. S
S VHS
VHS
VHS
VHS
~ S VHS --VHS
.
VHS
S
.
.
Dogfish 21 S Salmonides 17"7 S
---
HS
. HS
--
S --HS ~ S
--
-. VHS
Dogfish 21 S Lungfish 22"5 S
.
-HS
--
--
--
HS S ----
HS H S
S
--
Crocodile 18"6 S Python 19"5 S
HS, highly significant, 1
S, s i g n i f i c a n t , c l o s e t o P = 5 p e r c e n t . S, s i g n i f i c a n t , 5 < P < 1 per cent.
sedimentation coefficients of various thryoglobulins studied. values obtained for ½Cys were not near enough to allow for a statistical calculation.
S
Leu
* The ]" T h e
. --
Met Iso
--S ---
Ser Glu Pro Gly Ala
S
-.
Asp Thr
½Cys'~ Val
--S
acids
Lys His Arg
Amino
Eel 19"2 S Salmonides 17"7 S
Species
.
-HS
VHS
VHS
VHS
VHS HS HS HS VHS
S VH S
S --
--
--
VHS
S -----
-H S
S VHS --
Lungfish+ Dogfish
Salmonides VHS VHS VHS
Reptiles
Reptiles
ANALYSIS OF A M I N O ACID C O M P O S I T I O N S OF VARIOUS L O W E R VERTEBRATE T H Y R O G L O B U L I N S S T U D I E D
Trout 17"7 S * Salmon 17-7 S
TABLE 3--VARIANCE
t~ ~0
z
z
©
O
z
>
5"4
7"5
5"4 9-4 12.8 7.8
Arg
Asp
Thr Ser Glu Pro
9-8 1"7 4"6
1"3 2-5
1-8 7-5
7.7 7"9
5"9 8.7 12.7 7"2
8"8
5"9
1"7
3"4
Eel
9-3 2"4 5"3
1-2 4"2
2"1 6"8
7.8 6.0
6"2 8.5 12.1 6-5
9"5
4"5
2"1
4"6
Dogfish
8-3 2-3 5"4
1-3 3"9
3"4 7"2
6.8 5-2
6"7 11.0 12.1 5.6
9-7
3"5
2"1
4"8
Lungfish
9"3 2-5 5-9
1"8 4"2
2"4 6"2
7.3 5"7
5"7 9.7 11.7 5.7
10"5
4"0
2"3
5"0
Xenope
8-9 2"3 5"3
1"1 4-0
4"0 5"5
7"9 5.8
7"0 9-0 12-8 6.1
9-7
4"4
1"1
4"7
Python
9"5 2"3 5"6
1"3 4"4
3-1 6"4
7"6 6-1
5"8 5.0 13-7 7.2
11-0
5"0
1"4
4-9
Crocodile
9"2 3"4 5-3
1"5 3"8
4"0 6"1
7"9 6.1
5"2 10-1 13"5 5-3
9"2
4"7
1"1
3-9
Duck*
8"9 3"2 5-5
1"3 4"2
2"6 5-8
7"3 6"3
5"3 8.9 12.2 9.7
9"6
4"2
1"1
3"7
Chickent
9"2 2"1 5"0
0"9 2"4
4"1 6"5
7.5 8-6
5"0 10.0 12"8 7"9
6-8
6"4
1"3
2"7
Ox
Results are expressed as moles/100 moles of total amino acids. t Values reported by Hoshino & Ui (1970). * Values reported by Rolland et al. (1966). Mean of values obtained by Pierce et al. (1965), Rolland et al. (1966), Hoshino & Ui (1970) and Spiro (1970).
9"9 2-0 3"7
10-3 1"9 3"8
Tyr Phe
Leu
1"6 2"1
1-3 2"0
Met Ile
3"6 7.5
2-3 8"1
7.7 7"4
5"8 9.7 13.1 6.8
7"7
5"2
2-0
3'2
Salmon
Species
A M I N O ACID COMPOSITIONS OF PURIFIED THYROGLOBULINS OBTAINED FROM VARIOUS ~ R T E B R A T E CLASSES
~Cys Val
Ala
7"9 8.1
2"0
His
Gly
3"1
Lys
Amino acids T r o u t
TABLE 4
9-2 2"1 5"0
1"3 2"9
3-8 6-1
7"6 7-4
5"3 9-0 13-3 6-5
7"6
5"4
1"4
3"5
Humans
L/I ".-.I
0
Z o
8
0
>
O
58
A. BRISSON,J. MARCHELIDONANDF. LACHIVER
whereas it is very differentfrom salmonidthyroglobulins, since the contents of all amino acids except tyrosine differ significantly.
2. Comparison of the amino acid composition of thyroglobulinsfrom different vertebrate classes The results obtained for various thyroglobulins studied and listed in Table 4 are expressed as moles/100 moles of total amino acid recovered, in order to compare them with those given by other authors (Rolland et al., 1966; Hoshino & Ui, 1970) for the thyroglobulins of one amphibian (Xenopus laevis), two birds (Gallus gallus; Anas platyrhynchos) and one mammal (Homo sapiens). It is seen that a noteworthy degree of similarity exists between the thyroglobulins of xenope, python, crocodile, chicken, duck and those of dogfish and lungfish. The amino acid compositions of all these vertebrate thyroglobulins differ widely from those of teleost and mammalian thyroglobulins, especially for the content of Lys, Arg, Asp, Pro and Ile. The teleost thyroglobulins are relatively homogeneous. Their amino acid compositions are closer to mammalian thyroglobulins, from which they differ chiefly for the valine and phenylalanine contents than that of the other fish thyroglobulins studied (lungfish and dogfish). Considering all the thyroglobulins together, approximately 50 per cent of the total residues are aliphatic amino acids, while the basic amino acids account for 10-11 per cent and the dicarboxylic amino acids make up 20 per cent of amino acid residues. It is of interest that, although the contents of each basic amino acid vary widely according to the species studied, the total amount of basic residues differs little. DISCUSSION Several immunological studies, especially on mammalian thyroglobulins, have provided evidence about the species specificity of thyroglobulin (Hecktoen et al., 1927; Adant & Spehl, 1934; Stokinger & Heidelberger, 1937; Yagi & Kodama, 1955; Martinelli et al., 1969; Hoshino & Ui, 1970). The quantitative precipitin analysis and double-diffusion tests have. indicated that mammalian thyroglobulins are more or less related immunologically to each other, but no cross-reaction has been shown between mammalian thyroglobulins and those from two other vertebrate classes, Aves and Amphibia (Hoshino & Ui, 1970). For our part, the existence of significant differences between the sedimentation coefficients of fish thyroglobulins (Lachiver et aL, 1966) suggested possible species variations in the structure of these proteins and fitted in with the hypothesis of thyroglobulin specifieity. On the other hand, we have determined the amino acid composition of thyroglobulins from various lower vertebrate classes (Chondrichthyes, Osteichthyes, Reptilia).
AMINO ACID COMPOSITION OF THYROGLOBULINS FROM VERTEBRATES
59
The amino acid analyses of several mammalian thyroglobulins have been performed by different authors in recent years (Pierce, 1965; Rolland et al., 1966; Spiro, 1970; Hoshino & Ui, 1970), but hitherto the non-mammalian thyroglobulins have been the subject of very few studies: only the thyroglobulins from one amphibian and two birds have been investigated by Rolland et aL (1966) and Hoshino & Ui (1970). These proteins have been found to be rather different from those of mammals. In the present work, amino acid analyses of thyroglobulins from various fish and reptile species have indicated some similarity in the general composition of these purified proteins with thyroglobulins from other vertebrate classes. These results thus provide evidence of the homology (Florkin, 1962) of the major thyroidal protein in the whole zoological gnasthastome vertebrate series. However, these homologous proteins are not identical. Indeed a detailed examination of data obtained (Tables 2 and 3) has displayed the existence of significant interspecies variations in the contents of numerous amino acids and especially of basic (Lys, Arg), acid (Asp) and hydrophobic (Pro, Ala, Ile, Leu, Phe) residues. A variance analysis of preliminary results (Marchelidon et aL, 1972) has suggested that the more different the sedimentation coefficient thyroglobulins, the more distinct the amino acid composkions of these proteins. Yet, a comparison of data concerning the thyroglobulins from different vertebrate classes has shown that the importance of amino acid variations is independent of sedimentation coefficient differences. Indeed a great similarity of amino acid composition has been observed between the amphibian, reptile and bird thyroglobulins, having sedimentation coefficients close to 19 S, and the dogfish and lungfish thyroglobulins, whose sedimentation coefficients were definitely higher than 19 S (i.e., 21 and 22.5 S respectively). It is possible that, despite similar composkions, these various protein molecules could present variable amino acid sequences and therefore distinct conformational structures. Nevertheless, as in the case of other classes of homologous proteins, the ratios of hydrophobicity to fractional charge calculated, as reported by Welscher (1969), for the various thyroglobulins investigated, fall within very narrow limits, and the total number of hydrophobic amino acids is nearly constant. These data argue for close secondary and tertiary structures (Rolland et aL, 1966). On the assumption that these various proteins are not too different in shape and have partial specific volumes which are approximately identical, the hypothesis of molecular weight variations can be suggested. A crude estimate of the apparent molecular weight (mw) of various thyroglobulins can be obtained from the formula of Schachman (1959): mw2_/S~, mwl \ S J where mWx and S x are the molecular weight (670,000) and sedimentation coefficient (19 S) of ox thyroglobulin. For example, lungfish and salmonid thyroglobulins would have a molecular weight of 860,000 and 600,000 respectively.
60
A. BRISSON, J. MARCHELIDON AND F. LACHIVER
Would the number of polypeptide subunits of this protein change in some species ? But as long as the number and size of thyroglobulin subunits are a controversial subject (see Bornet, 1971; Rolland & Lissitzky, 1972) no serious interpretation of our data can be given. On the other hand, in considering the evolutionary tree of vertebrates (Fig. 1), thyroglobulin amino acid compositions seem to be connected with phylogenetic relationships between the various animal species investigated.
/ (Pyth0n,Croc0dite)
HOLO$TEI
/
CROSSOPTERY611 / >OlPflOI (Lungfish)
CHONDR(]~
ACTINOPTERYGil , CHOAHICHTYES \ v /
"
/
/
CHOHpRIGHTHYE$ ~ (Dogfish)
Fig:1 A simplified pbylogenetictree of the gnathostomevertebrates outlined from ROMER(1949) and GRASSE(1958) * 1he namesof vertebrate classesare underlined It can be seen that the composition of dogfish and lungfish thyroglobulins are closer to amphibian and reptile thyroglobulins than to that of other fishes such as the teleosts, but the forms arranged in the Pisces superclass belong in fact to three independent evolutive lines: the Chondrichthyes, the Actinopterygii and the Choaniehthyes, the two end-branches of which are represented by the classes of Aves and Mammalia. Table 4 shows that the various thyroglobulins studied can be classified into three groups: teleost, mammals and other vertebrates. From a phylogenetic point of view, the evolution of the structure of different proteins having a lower molecular weight (such as hemoglobin, cytochrome, insulin) has been studied (see Acher, 1957; Neuman & Humbel, 1969; Jukes, 1971; Diekerson, 1971). Substitution and duplication events would be essential in protein evolution and could breed several evolutive lines from a common ancestral molecule. From these data, it may be supposed that thyroglobulin composition would have varied independently in the Actinopterygii and Mammalia branches of
AMINO ACID COMPOSITION OF THYROGLOBULINS FROM VERTEBRATES
61
the evolutionary tree, whereas it has remained closer to the composition of the ancestral molecule in the Chondrichthyes, Choanichthyes, Amphibia, Reptilia and Aves. SUMMARY 1. Thyroglobulins of five fishes (trout, salmon, eel, dogfish, lungfish), two reptiles (python, crocodile) and one mammal (ox) were purified. 2. The amino acid composition of these purified thyroglobulins has been carried out. A comparison of results obtained with thyroglobulins from other vertebrate classes has shown the homology of the thyroglobulin throughout the series of vertebrates, from lower vertebrates to mammals. 3. However, a variance analysis has pointed out very significant variations of the numerous amino acid contents (especially Arg, Lys, Asp, Pro, Ala, Ile, Leu and Phe). 4. The importance of amino acid composition variations is independent of sedimentation coefficient differences of the thyroglobulins studied. 5. On the contrary, the variations of amino acid compositions seem to be correlated to the phylogenetic relationships of the species investigated. Acknowledgements--The authors wish to express their thanks to Miss Boulu Franqoise for her valuable technical assistance.
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63
WE~Cm~R H. D. (1969) Correlations between amino acid sequence and conformation of immunoglobulin light chains--I. Hydrophobicity and fractional charge. Int. J. Prot. Res. 1, 253-265. YAGI Y. 8z KODAMA M. (1955) Etudes immunochimiques des prot6ines thyroidiennes. C. R. Soc. Biol. 149, 2282-2284. Key Word Index--Thyroglobulin; amino acid composition; lower vertebrates; phylogenetic relationships of thyroglobulins; thyroglobulin specificity; sedimentation coefficient thyroglobulin.