Evolution of Gonadotropin Molecules

Susumu IshiiDepartment of Biology, Waseda

University, Tokyo 169-8050susumu@mn.waseda.ac.jp

Do LH, FSH and CG exist in all vertebrate species?

LH FSH CGMammals Yes Yes Yes or no

Birds Yes Yes No

Reptiles Yes Yes No

Amphibians Yes Yes No

Lungfishes ? Yes No

Bony fish GTH-II GTH-I No

Cartilage fish ? ? No

How do gonadotropin

molecules look like, a three dimensional

model constructed

with RasMol (a computer program)

How are gonadotropin molecules different among hormone species within a species?

1) β subunits are hormone specific.2) α subunits are identical.

FSH LH TSH

α αα

β β β

α

β

CG

α

β

Dimeric structure of ganadotropins

Identity values (%) between human gonadotropin β subunit molecules

LH FSH

CG

32.8%

66.9%

26.0 ％

ＴＳＨ ＴＳＨ

38.4% 38.3%

29.3%

Two presumed models of LH and FSH differentiation

FSH FSHLH LH

A B

Percent identities between LH and FSH β subunit molecules in various

vertebrates

Animal group No. of species Mean identity S.E.M.Amphibians 2 39.6 1.2Reptile 1 38.6 - Birds 2 34.6 1.7Mammals 3 32.5 0.5

Conclusion

The difference in the primary structure between LH and FSH β subunit molecules becomes larger as the animal becomes higher. The Model B is correct.

Gonadotropin subunits

1) LH β subunit

2) FSH β subunit

3) CG β subunit

4) (TSH β subunit)

5) Common α subunit

How are gonadotropin subunits different among vertebrate

species?

Difference in the primary structure

Com m o n a lp h a

74

81

88

98

100

To a d

Tu r t le

Qu a il

Ra t

Po r c in e

LH be t a

39

44

43

80

100

To a d

Tu r t le

Qu a il

Ra t

Ov in e

FSH b e t a

56

67

66

86

100

To a d

Tu r t le

Qu a il

Ra t

Ov in e

Evolution speed differs between subunit and hormone species.

Fastest: LH βMedium: FSH β

Slowest: Common α

How are gonadotropin subunits different among vertebrate

species?

Secondary structure

LH : : : : : : : :1::: : : : : :2::: : : : : :3::: : : : : :4::: : : Bovine ooooiooiiooioHHHHHHHooioiiEEEEEEEEEEEiiooooiEEPig ooooiooiiooioHHHHHHHooEEEEEEEEEEEEEEEiiooooioiChicken ooooiooiiooioHEEEEHHooioiiEEEEEEiiEEEiiooooioiBullfrog ---oio--iooioioii iooooEEEEEEEEEEiiEEEiiooooiioFSHBovine ------oiiooioiEEEEHHHHiEEEEEEEEEEEiiEEEooooiioRat ------oiiooioiEEEHHHHHiEEEEEEEioEEEEEEEooooiioOstrich -------- iooioioHHHHHHHHHHiii ioioEEEEEEEooooiioBullfrog -------- iooioiEEEEooooioEEEEEoioEEEEEEEooooiio

H:α helix E: β sheet i & o: loop

LH 7::: : : : : :8::: : : : : :9::: : : : : :0::: : : : : :1::: : Bovine EEooioooiooEEEEEEEEEioiooioiooooioiooiEE-EEEEPig EEooioooiooEEEEEEEEEioiooioiooooioiooioo-oiioChicken EEEoioooiooEEEEEEEEEioiooioiooooioiooioo-oiioBullfrog EEooioooiooEEEEoiiioioiooioiooooioiooioo-oiioFSH 7:: :: : : : :8:: :: : : : :9:: : : : : : :0:: : : : : : :1:: : : Bovine EEooioooioEEEEEoiiiEEEEEoioiooooioiooioo-oiioRat EEooioooioEEEEEoiiiEEEEEoioiooooioiooioo-oEEEOstrich EEooiHHHHHHHEEEEiiiEEEiooioiooooioiooioo-oEEEBullfrog EEooiHHHHooEEEEEEEEEioiooioiooooioiooioo-oEEE

H:α helix E: β sheet i & o: loop

large loop

N-terminus of alpha

N-terminus of beta

beta-chain

alpha-chain

C-terminus of alpha

C-terminus of beta

determinant loop

signal transduction

FSH/LH specificity

FSH/LH specificity

Model of the gonadotropin molecule

Model of the gonadotropin molecule

Model of the gonadotropin molecule

Conclusion

The primary structure has diver ｇ ed during the course of the evolution, but the secondary and tertiary structures and hence the three dimensional structure ha ｓ been conserved.

Evolution of the common α subunit in vertebratesfrom fish to mammals

60 70 80 90 MLVPKNITSEATCCVAKSFTKATVMGNERVENHTDCHCSTCYYHKSMLVPKNITSEATCCVAKAFTKATVMGNARVENHTECHCSTCYYHKSMLVPKNITSEATCCVAKAFIRVTLMGNIRLENHTQCYCSTCYHHKLMLVQKNVTSESTCCVAKSYNRVTVMGGFKVENHTACHCSTCYYHKSMLVPKNITSEATCCVAKAFTKITLKDNVKIENHTDCHCSTCYYHKSMLVPKNITSEATCCVAKEVKRVLV.NDVKLVNHTDCHCSTCYYHKSMLVPKNITSEATCCVAKEFKQVLV.NDIKLVNHTDCHCSTCYYHKSMLVPKNITSEATCCVAKEGERVVV.DNIKLTNHTECWCNTCYHHKSMLVPKNITSEATCCVAKEGERVTTKDGFPVTNHTECHCSTCYYHKSMTIPKNITSEATCCVAKHSYETEV.AGIKVRNHTGCHCSTCYFHKIMLVPKNITSEATCCVAREVTRL...DNMKLENHTDCHCSTCYYHKF

10 20 30 40 50LPDGDLIIQGCPECKLKENKYFSKLGAPIYQCMGCCFSRAYPTPARSKKTFPDGEFTMQGCPECKLKENKYFSKLGAPIYQCMGCCFSRAYPTPARSKKTFPDGEFTTQDCPECKLKKNKYFSKLGVPIYQCMGCCFSRAYPTPARSKKTAPD....VQDCPECTLQENPFFSQPGAPILQCMGCCFSRAYPTPLRSKKTFPDGEFLMQGCPECKLGENRFFSKPGAPIYQCTGCCFSRAYPTPMRSKKTYPRNDMNNFGCEECKLKENNIFSKPGAPVYQCMGCCFSRAYPTPLRSKKTYPRNYMNNFGCEECKLKENNIFSKPGAPVYQCMGCCFSRAYPTPLRSKKTYQNSDMTNVGCEECKLKENKVFSNPGAPVYQCTGCCFSRAYPTPLQSKKAYPNSDKTNMGCEECTLKPNTIFPN....IMQCTGCCFSRAYPTPLRSKQTYPSMDLSNMGCEECTLRKNSVFSRDR.PVYQCMGCCFSRAYPTPRKVMKTYPNNEMARGGCDECRLQENKIFSKPSAPIFQCVGCCFSRAYPTPLRSKKT

RatPorcineDonkeyHuman

QuailCarp 1Carp 2

Salmon 1Salmon 2

Striped bassEuropean eel

RatPorcineDonkeyHuman

QuailCarp 1Carp 2

Salmon 1Salmon 2

Striped bassEuropean eel

43%(64%)

60 70 80 90 ---------------------KATV-GNERV----------------------------------A--KATV-G-ARV----E----------------------S------A----AV-G-I-L----Q-Y-----H--I----Q--V---S-------YN--TV-GGFKV----A-----------------------------A--KITLK--V------------------

-----------------------A-----------------------TMLVPKNITSEATCCVAKSFTRVILMDNLKIENHTDCHCSTCYYHKS

------------------EVK--LV.NDV-LV---------------------------------E-KQ-LV.NDI-LV---------------A-----------------EGE--VV.--I-LT---E-W-N---H---------------------EGE--TTK-GFPVT---E-------------TI--------------HSYETEV.AGI-VR---G-------F--I-----------------REV--L...--M-L---------------F

10 20 30 40 50L-DGDLII-.--------------KL-------M-----------A------DG--TM-.--------------KL-------M-----------A------DG--TT-.D------R-----FKL-V-----K-----------A--R-A-D....V-.D----T-Q--PF--Q-----L--M-----------L------DG--LM-.-------G--RF--K--------T-----------M----

--D--LI--A--------------K--------S-----------A----FPEEEFMAQVGCPECKLKENKYFSRPGAPIYQCAGCCFSRAYPTPVRSKK

Y-RNDMNNF.--E-------NI--K----V---M-----------L----Y-RNYMNNF.--E-------NI--K----V---M-----------L----YQNSDMTNV.--E--------V--N----V---T-----------LQ---Y-NSDKTNM.--E--T--P-TI-PN....-M--T-----------L---QY-SMDLSNM.--E--T-RK-SV---DR.-V---M-----------RKVM-Y-NN-MARG.--D--R-Q---I--K-S---F--V-----------L----

RatPorcineDonkeyHuman

Quail

Australian lungfishAfrican lungfish

Carp 1Carp 2

Salmon 1Salmon 2

Striped bassEuropean eel

RatPorcineDonkeyHuman

Quail

Australian lungfishAfrican lungfish

Carp 1Carp 2

Salmon 1Salmon 2

Striped bassEuropean eel

Human73 Rat72 　 92 Porcine68 82 85 Donkey70 80 84 76 Quail71 85 83 78 84 Australian lungfish70 79 80 76 83 92 African lungfish68 73 71 67 74 73 72 Carp 167 73 72 68 74 73 72 96 Carp 260 66 67 65 66 68 68 77 76 Salmon 164 64 65 60 64 63 63 69 67 72 Salmon 256 60 58 57 56 57 57 64 64 61 60 Striped bass65 69 69 66 72 73 72 76 74 68 65 56 European eel

97 64

94

38

76

56

Rat

European eel

Striped bass

Salmon

Carp

Australian lungfish

African lungfishQuail

Donkey

Porcine45

60 70 80 90 ---------------------KATV-GNERV----------------------------------A--KATV-G-ARV----E----------------------S------A----AV-G-I-L----Q-Y-----H--I----Q--V---S-------YN--TV-GGFKV----A-----------------------------A--KITLK--V------------------

-----------------------A-----------------------TMLVPKNITSEATCCVAKSFTRVILMDNLKIENHTDCHCSTCYYHKS

------------------EVK--LV.NDV-LV---------------------------------E-KQ-LV.NDI-LV---------------A-----------------EGE--VV.--I-LT---E-W-N---H---------------------EGE--TTK-GFPVT---E-------------TI--------------HSYETEV.AGI-VR---G-------F--I-----------------REV--L...--M-L---------------F

10 20 30 40 50L-DGDLII-.--------------KL-------M-----------A------DG--TM-.--------------KL-------M-----------A------DG--TT-.D------R-----FKL-V-----K-----------A--R-A-D....V-.D----T-Q--PF--Q-----L--M-----------L------DG--LM-.-------G--RF--K--------T-----------M----

--D--LI--A--------------K--------S-----------A----FPEEEFMAQVGCPECKLKENKYFSRPGAPIYQCAGCCFSRAYPTPVRSKK

Y-RNDMNNF.--E-------NI--K----V---M-----------L----Y-RNYMNNF.--E-------NI--K----V---M-----------L----YQNSDMTNV.--E--------V--N----V---T-----------LQ---Y-NSDKTNM.--E--T--P-TI-PN....-M--T-----------L---QY-SMDLSNM.--E--T-RK-SV---DR.-V---M-----------RKVM-Y-NN-MARG.--D--R-Q---I--K-S---F--V-----------L----

RatPorcineDonkeyHuman

Quail

Australian lungfishAfrican lungfish

Carp 1Carp 2

Salmon 1Salmon 2

Striped bassEuropean eel

RatPorcineDonkeyHuman

Quail

Australian lungfishAfrican lungfish

Carp 1Carp 2

Salmon 1Salmon 2

Striped bassEuropean eel

The reason why the lungfish α subunit is more similar to tetrapod vertebrates than

to ray-finned fishes

1) Lungfish are phylogenically closer to higher tetrapods than to ray-finned fishes

2) Ray-finned fishes are phylogenically far higher animals than generally believed

If the assumption 1) is correct,

lungfish must be closer to tetrapod than we thought.

If the assumption 2) is correct, lungfish must be closer to elasmobranchs (sharks and rays) or elasmobranchs are closer to tetrapods than we thought.

60 70 80 90-------------------S-TKA-V---ERV----D------------------------------A-TKA-V---ARV-----------------------------------A-I-------IRL----Q-Y-----H--I-----Q------S------SYN---V--GF-V----A------------------------------A-TKI--KD-V------D------------------------------A-TKI--KD-V------D------------------------K-----TQY---V-D-V------A------L-----------------K-----TQ----I.NGM------A------L---F-------------------T-T--SHLDRMN-----D------------------------------S-T--A--D--------D------------------------------S-T--I--D--------D-----------

KTMLVPKNITSEATCCVAKLFSRVTLMGNLKIENHTECHCSTCYYHKS

-------------------EVK--LV.NDV-LV---D------------------------------E-KQ-LV.NDI-LV---D------------A-----------------EGE--VV.D-I-LT-----W-N---H---Q------------------EGE---TKDGFPVT------------------TI--------------HSYETEV.AGI-VR---G-------F--I------------------REVT-L...D-M-L----D----------F

RatPorcineDonkeyHuman

QuailTurtle

BullfrogToadNewt

Australian lungfishAfrican lungfish

Smooth dogfish

Carp 1Carp 2

Salmon 1Salmon 2

Striped bassEuropean eel

Human73 Rat72 　 92 Porcine68 82 85 Donkey70 80 84 76 Quail70 80 83 77 99 Turtle66 69 71 68 74 75 Bullfrog63 63 65 65 67 67 80 Toad64 73 76 72 76 75 70 69 Newt70 79 80 76 83 83 73 69 77 African lungfish71 85 83 78 84 84 74 68 76 92 Australian lungfish68 72 75 73 75 75 74 73 76 80 78 Smooth dogfish68 73 71 67 74 74 66 64 67 72 73 67 Carp 167 73 72 68 74 74 64 63 67 72 73 67 96 Carp 260 66 67 65 66 66 62 57 60 68 68 63 77 76 Salmon 164 64 65 60 64 64 62 61 62 63 63 65 69 67 72 Salmon 256 60 58 57 56 56 54 53 56 57 57 55 64 64 61 60 Striped bass65 69 69 66 72 72 63 65 69 72 73 69 76 74 68 65 56 European eel

Conclusion

The α subunit of ray-finned fishes has evolved more highy than we thought,

and the evolution speed of the α subunit in the ray-finned fishes should be faster than that in the other vertebrate groups.

Summary1) During the course of the evolution, the

primary structure has diverged much but the higher order structures have been conserved.

2) The evolution speed differed among the subunit molecules, being fastest in the LH β , medium in the FSH β and slowest in the common α .

3) The evolution speed of the α subunit differed among vertebrate groups, faster in the ray-finned fishes than in the other groups.

Collaborators

Higher order structures: Prof. H. Wako

cDNA cloning: Drs. K. Kubokawa, H. Ando, M. Kikuchi, Y. Arai, Y. Komoike and Y. Aizawa.

Lungfish study: Prof. J. Joss