Kanehisa, Minoru

Seto, Yasuhiko

Kyoto University Research Information Repository

TitleRepeat Sequences of Amino Acids Suggest the Origin ofProtein (Commemoration Issue Dedicated to Professor TatsuoOoi, On the Occasion of His Retirment)Author(s)Seto, Yasuhiko; Kanehisa, MinoruCitationBulletin of the Institute for Chemical Research, KyotoUniversity (1989), 66(4): 461-468Issue Date1989-02-28URL http://hdl.handle.net/2433/77258RightType Departmental Bulletin PaperTextversionpublisherKyoto UniversityBull. Inst. Chem. Res., Kyoto Univ., Vol. 66, No. 4, 1989        Repeat Sequences of Amino Acids Suggest                the Origin of Protein                 Yasuhiko SETO* and Minoru KANEHISA**                             Received November  1, 1988    In order to test our oligoglycine hypothesis for the generation of primordial proteins, we examined amino acid sequence repeats in present proteins stored in the PRF/SEQDB database. We found high occurrences of glycine containing repeat patterns such as GX, GGX, GGGX, GGXY, GGGGX, GGGXY, where G is glycine and X and Y are other amino acids. This is consistent with our hypo-thesis that short oligoglycines reacted with aldehydes and alkenes to form various oligopeptides as basic building blocks of protein molecules.    KEY WORDS: Oligoglycine Hypothesis/ Repeat Sequence/ Molecular Ev-                   olution                          I. INTRODUCTION    The earth was thought to be born 4.6 billion years ago. Since then it took 0.6 billion years for the creation of life. The first 0.6 billion years and the following 4 billion years seem to have principally different meanings for proteins. Before the appearance of life primordial forms of proteins may have been produced. There are several hypotheses concerning the production of amino acids and nucleotides. Ohno's idea is an interesting one which states that nucleic acids have evolved from oligonucleotides by duplication (1). Akabori proposed that polyglycine is the pri-mordial protein and it reacted with aldehydes and unsaturated compounds to form polypeptides containing various amino acids (2). Akabori's hypothesis explains well how the amino acids of proteins have been produced and why the amino acids are alpha amino acids. However, it is unlikely that functional proteins containing tens to hundreds of amino acids were derived directly from polyglycines as Akabori originally proposed. It is hard to imagine that various reactions occurred at various locations on a long polyglycine chain. In fact, it is simpler to think that short oligo-glycines reacted with aldehydes and alkenes as in Akabori's hypothesis to form vari-ous oligopeptides. Short oligopeptide chains may condense with each other to form long protein chains. The idea implies that the structural unit of a protein is an oligopeptide. With this hypothesis in mind we are trying to find a clue for the evolution of protein molecules. In this report we analyze amino acid sequence repeats in present functional proteins.                    II. METHODSOF CALCULATION    For the analysis of amino acid sequences we used version 87.5 of the protein *p : Protein Research Foundation, Peptide Institute, Ina, Minoh, Osaka 562 ** A : Institute for Chemical Research, Kyoto University, Uji, Kyoto 611 (461)                                               Y. SETO and  'M.'KANEHISA          sequence database of the Protein Research Foundation (PRF/SEQDB). The data-          base SEQDB has been produced by the Protein Research Foundation since 1979,           along with the literature database LITDB and its printed version "Peptide Informa-         tion". The PRF/SEQDB database contains full and partial sequences of natural          peptides and proteins with more than 50 amino acid residues. PRF/SEQDB ver-          sion 87.5 contains the sequence data extracted from literature published before          December 1986, totaling 2,351,308 residues in 10,021 entries.              An amino acid sequence is represented by 20 letters: A(Ala), C(Cys), D(Asp), E(Glu), F(Phe), G(Gly), H(His), I(Ile), K(Lys), L(Leu), M(Met), N(Asn), P(Pro), Q(Gin), R(Arg), S(Ser), T(Thr), V(Val), W(Trp), and Y(Tyr). We searched re-         petitions of all possible amino acid patterns with lengths 1 through 15. An amino           acid sequence repeat is characterized by two indices n and m, where n is the length           of an oligopeptide repeat unit and m is the number of repetitions. We searched          both continuous and discontinuous repeats against the PRF/SEQDB detabase. Re-          peat units are perfectly aligned in continuous repeats, while repeat units may be           separated by any number of other amino acids in discontinuous repeats. We ex-          tracted continuous repeats with the following threshold values for the minimum           number of repetitions: 4 for n=1, 3 for n=2, and 2 for n=3-15. The threshold values for discontinuous repeats were: 6 for n=3, 4 for n=4, 3 for n=5, and 2 for            n=6-15.                                      In. RESULTS           3.1. Amino acid composition              The average amino acid composition in all the entries of PRF/SEQDB was the following:           L 9.10%, A 7.55%, G 7.35%, S 7.20%, V 6.45%, E 6.06%, K 5.94%,           T 5.92%, P 5.18%, R 5.15%, I 5.15%, D 5.13%, N 4.31%, Q 4.17%,           F 3.96%, Y 3.30%, H 2.30%, M 2.24%, C 2.09%, W 1.38%          Thus, glycine was the third most abundant amino acid component. When the          composition was examined individually, 1,009 entries out of 10,021 had glycine as           the most abundant amino acid component, while 2522 and 1099 entries, respectively,           had leucine and serine as most abundant.           3.2. Repeats of single amino acids              We first searched continuous repeats of single amino acids with four or more           residues. The longest repeat for each amino acid was:          P 29, E 27, G 20, Q 19, N 15, A 14, S 14, D 13, H 14, R 11,          L 10, K 8, T 7, F 7, I 6, V 5, Y 5, C 4, M 4          Repeats of 4 or more residues were not found for tryptophan. Repeats of up to           only 4 residues were found for cystein and methionine. Proteins with long repeats           of single amino acids were as follows: ( 462 )                      Repeat Sequences of Amino Acids Suggest the Origin of Protein           P 29: Epstein-Barr virus protein          E 27: high mobility group protein HMG           G 20: Ca dependent protease S  Q19: glucocorticoid receptor        The total number of occurrences of repeats with four or more residues for each amino        acid was:       L 420, P 279, R 259, A 250, S 250, G 190, E 177, Q 164, K 88, N 65,        T 63, D 52, H 41, V 36, I 16, F 13, C 13, Y 4, M 2, W 0        Thus, repeat sequences appeared frequently for eight amino acids: leucine, proline,        arginine, serine, alanine, glycine, glutamic acid, and glutamine. The proteins which        were mainly composed of repeating amino acids or rich in specific amino acids were: S: phosvitin            A: antifreeze protein           H: Plasmodium antigen            G: petunia structural protein       3.3 Repeats of oligopeptide units          Repeating oligopeptide units of sizes 2-15 were identified from the PRF/SEQDB        database with the threshold values given in the Methods section. The results are       shown in Tables I—III. Table I summarizes the patterns found for dipeptide repeats.                                Table I. Dipeptide repeat patterns found 2nd             G SPED AVKR LQT F Y NH I CMW 1 st  G + + + + + + + + + + ++ + S + + + + + + + + + + + + P + + + + + + + + + + E + + + + + + + + ++ D + + + + + + + + + +    A + + + + + + + + +   ^ + + + + +++ + K + + + + + + + + R + + + + + + + + L+ + + + + + + + Q + + + ++ + T + + + ++ F+++ + + Y+ ++ + N+ + ++ H + + ++  I+ ++ C ++ M W (463)                         Y.SETO and M. KANEHISA                 Table II. List of tripeptide repeat patterns found                 XYZ 七ype                           XXY 七ypeACG   AML   CPK   DTN   FLG   GSK   KTS   PUT   GGA   EEA   RRA   YYG  R     Q   .CQK     V      l     P   KVS   PYQ     ・C     D     F      lADGRLDVLRRTQSTDFGT  L.ANC CRL T S Y LNR Y E L K CCGAEG     L   CSN   DWS   FPG   GTP   LPQ   QYS     F     P     L     S  K   APE   DEL   DYE     R     S     R   RTV     I     Q     M   NNI  L     G     P     N   FSK   GVI     S   STV     K     R     S     Y  PRDFIEFQMLV  LST-FFS  4   A4K     V   EGL     V     M   L4N          M     T    ・V  R     L   DGI   EIK   FTL     N     P           N     V   PPA  V   ARF     K     Q   FUL     P     T           P   SSA     GAFL     I     L   EKG'  FVR   GWN     Y           S     E     KAGD     lく    N     L   GDV   GYS   LRS          V・   F     L  EしPEしFGHPHQPV  YGR  L V R GGIL HUL LSM  AAD L S  NASCDILKPIKNP  ENT  PLPQTQT  FPV  R     4     S     R   GKI     S   LTP          G     R   TTA  RRVSLVS  ITL  T     T   DKF     T     N   ILS     W           K     V     Q  V V GEML P T LUP   LQQA SAHRATG LENLGLP Y R  NDVAIEKQEPGQINKS  PEY  G     P   DLK     L     R     R   LYN          Q     F   DDE  KRRF.QLSIPLMFT  R.GK  L S S N TIQL MST  S I M  Q      V      U      P   GML    IRS   MYN            T      L      TAKE AVE Y ERG P ISK R   V M Y  R    G   DNS    L    V     L   NST        LLA    P   VVA  S KDPE S GNS R VI  DKKA G'  V    L    I  ESL   GPK   ITN   NTQ         E    E    LALDPLRLSS  FGR  E     Q     R   ETW     Q   IVN     U          G     L   HHA  G     S   DQE   EVL     R   IYV   NVS            I     M     P  H   AWQ'    G   EYG    S   KLM   NYT          M    P    T  K  AVI    L  FGP    V    R  PRS        P.   R   IIG  N   CDK     S     S   GRI   KNS   PSR          S     S     T  Q   CGK   DRG   FHR     S     V     V          T     Y     V  R     L     L   FIV     T   KPQ   PT4          V  SPDSEFWH  SR  U   S   U          T   S                                (464)              Repeat Sequences of Amino Acids Suggest he Origin of Protein                   Table III. List of oligopeptide repeat patternsfound  4-PEPTIDE5-PEPTIDE6-PEPTIDE   G3A FILL GAGQ ANSL G4E GSGAA AVSQD G5L TPAPAA    F IGLL GLGV ARID F GGNPP CAERQ M TPATAA    K AGLL AGSG ASLP M AGAGG DLSTP E5D AEAAVD    L LLAA SGLG AVKN S CGCGG FNSTW G AHHAAD M LLVV AEAD AVPF A4P LGSGG RKYSI A5P AHHAAN    R LLCV ASAL CAER G3DT LGVGG SPRKG A4QQ DGVSKK    S ALLV IAQA DVLK SE LGSGG SPTKR E4VQ EPVTTQ    V GGSS KAKR EVTA SF DSGYGR4G2 GGYGGA V GGVP KAKS FIRK SS GQRAAG3MGM GO.GQQS   E3A DTGG CFIF FLND L3AS EPTCCGFG3S GQQPGQ    D SGGK DPDH FSLA WV DDEPVAGAG3 GSSAFA    G EEGQ EQDQ GQSR A3G2 EEGQQGGLS3 KEPTPP    I EERA FNFI IYLS N3SP EEKKDP3VHL KKDDEG   L EEKK GTFT KGLAFRLPPLAQQAS  A3E SSEE IYIS LDHVPHGHHLFNSTW AEGAPP   G EQKK LGLV LDIC1RRPRPGLPGS   K ETKK PKPA LSVYYLSIIGPAGPP   P TPKK NANP LTERAAKPKQQPFPP Q3E QQIL PQPH NKITKKPAAPRSPRE   P QQPF SIFI QGLNGVQNNPGQWQQ   R ELQQ WRWGQTLDNTNSSGGSVAS   S AAIG YIYL RAEPPASAATGSCVV  L3A SEAASLVQPPIHKPHQPLQ  F ESSTSPAFPLTRRPSPTAS  P RASSSPRKRRAWITGTAKV  R3G IIGLVCMYIYLSS H RRAKVSLYTCCPD  S31 LAFFWGENWIRRS T NNTSWPLGAEAKT  C3G TTNSWPVGAHHDG F3MRPERP I3C N3M  T3S The number of times a given amino acid occupied either or both of the positions in the dipeptide repeat unit was: G 13, S 12, P 10, E 10, D 10, A 9, V 8, K 8, R 8, L 8, Q 6, T 5, F 5, Y 4, N 4, H 4, I 3, C 2, M 0, W 0    Table II shows all the patterns in tripeptide repeats.The tripeptide patterns are classified into two types, those containing three different amino acids such as GCA, and those containing an internal repeat of one amino acid such as GGA. The usage of amino acids in the first type was determined, for example, by counting (465 ) Y. SETO and M. KANEHISA                            Table III. (continued)7-PEPTIDE 8-PEPTIDE 9-PEPTIDE 10-PEPTIDE R6C AEGAAPP AG7 GGCGCGCC H7DA AHHAHHVAD GH9 SYGGSSG4 S6K CGGCGCG D7E GGCGGCGC KPQGP4 CCGGCGGCG LQ9 MGMG3MG3 A4KRK NTTDNNT Q7L GGAGGAGA FG3YGGSS DRADGQPAG APH8 GGFGGRG3R SP4EH PQPQLPY R7C GGSGGRGR G3SVGGSS EQPAAGAGG DAH8 KPQGP3QGG SP4VH PQPQQPF R6CC PQPQPQPE G3SYG3S DRAAGQPAG CGCGCGGCGG PSTTIPA R4SQSP NANPNVDP G3S3GGY KKARKRPKC GTGTGTGSGS     PSSPSYS Q4PPFS EDNNKPGK GGAGAG3A NNVFQPSQQ HHDDAHHDGA     PTSPSYS G3LGAGF AKVTGTGTGQQGQQPGQ HKPPVYTPPV     PTSPNYS G3SGSGF KPGKEDGNSGDSEDKKE PEKRSKSGSR     PTSPKYS G3WGQPH KSDEAEALSGTSGTSAQ TSCCQPTSIQ    YGSGGSS N3MNHNM KSDEAEARAKVTGTGTGT     YGSGGGSREAAEQAK     LGYGGSSSPRKSPKK     KRFMRFGTPPTPSPS KRYGGFM     NEGLKTE SILEVIH the patterns of GXY, XGY, and XYG for glycine where X and Y represent two different amino acids. L 99, G 80, A 77, S 69, P 57, V 54, K 49, R 49, D 49, E 47, T 44, I 43, Q 41, N 32, F 25, Y 16, M 15, C 14, H 8, W 5, The total was 873 out of possible 20 x 19 x 18=6840. The patterns shown in Table II are unique patterns obtained by considering the difference in the phase at which the repeat unit begins; for example, XYZ, YZX, and ZXY are considered identical repeat nuits. The total number of unique patterns belonging to the first type was 291.    The usage of amino acids in the second type was determined by counting the patterns of GGX for glycine, for example. G 14, A 14, L 11, E 10, S 10, Q 9, K 9, R 9, P 8, T 6, D 5, V 4, H 3, I 3, Y 3, C 2, N 2, F 1, M 0, W 0 The total was 123 out of possible 20 x 19=380. It is therefore noteworthy that the second type containing the repetition of the same amino acid within a repeat unit appears to be relatively more abundant.    Table III shows repeat patterns of unit sizes 4-10 classified by the internal repeti-tion within each repeat unit. In 2 to 5-peptide repeat units the types of GX, G2X, G3X, G2X2, G4X, and G3X2 appeared to be more frequent, where in this notation X is any amino acid other than glycine and a numeral denotes the number of con-secutive runs. Types of X2Y, X3Y, and X4Y are also found many times, where X and Y are amino acids other than glycine. (466)                     Repeat Sequences of Amino Acids Suggest the Origin of Protein                                  IV. DISCUSSION          If primordial repeating units were produced from oligoglycines, we may find       repeat units with glycine as a basic component. This was our working hypothesis       in the present analysis.  In terms of possible mechanisms of producing different       amino acids, it is more probable that an oligoglycine is modified to give the same       amino acid at a few positions than to give all different amino acids as shown below:            GGGGG —k GGGXX more probable            GGGGG —* GABCD less probable      The abundance of repeat patterns of the types of GX, G2X, G3X, G2X2, G4X,       and G3X2 are, at least, consistent with the hypothesis.          Even if the production probability of repeat units containing different amino       acids are low, such units, once produced, may become important as functional units       to interact with various ligands such as other peptides, nucleotides, metal ions, sugars,       lipids, and so on. Examples of biologically active functional repeat units in present       proteins, which are extracted from the PRF/SEQDB database, are shown below:           peptide precursor size repeat sequence         FMRF amide309 KRFMRFGK (20 times)          enkephalin239 KRYGGXM (7 times, X=--F or L)         thyroliberin 255 KRQHPGXR (5 times, X=K or R)         mating factor alpha 165 WHWLQLKPGQPKR (4 times)       These functional units in proteins are often reported as common functional motifs or        conserved sequences.           Proteins with repeat sequences are found to be mainly structural proteins, storage       proteins, and viral proteins. These proteins may be considered primordial from       the standpoint of function. In dipeptide repeats, frequently appeared amino acids       are so called primitive amino acids, such as glycine, serine, and proline, which may       originate older than the other amino acids.           Biological organisms have developed various processes and systems for the ef-     ficiency and simplicity of their life during 4 billion years. Most significant is the       mechanism of constructing a protein molecule from amino acids following genetic       information. It appears less likely that polypeptides are produced by combining       amino acids one by one for each protein. Protein molecules may have evolved from       shuffling and combining of peptide segments. Recently there have been suggestions       that exons and modules are elementary units of proteins (3). Modules are defined       from the analysis of three-dimensional protein structures and usually composed of       about 20 amino acid residues, while the median of observed exon lengths is about       40 residues.          In addition to these structurally defined units, there are functionally important       units in the amino acid sequences. Many proteins have peptide fragments exhibit-      ing function of native proteins and sometimes activity which has no direct relation ( 467 )                                     Y.  SETO.  and. M. KANEHISA to mother proteins. Examples are shown below:   Peptide ProteinActivity    YGGFM beta lipotropin fragment morphine like activity   WMDF gastrin fragmenthormonal activity    RGDS fibronectin fragment cell adhesion activity    PTKKGKG nucleoplasmin fragment translocate to cell nucleus    A special region in a protein may have acquired one function during the long history of chemical evolution. Alternatively, a special oligopeptide with one func-tion may have grown up to a protein molecule adding various fragments for effi-ciency. If the latter process is the principal one, we may be able to find such pri-mordial functional unit peptides in the current proteins. We presume that they are composed of five to ten amino acid residues from the consideration of occurrence probability. We find present proteins to be usually multifunctional, but by analyz-ing repeating sequences we may be able to obtain insights into the character of pre-biotic proteins.                       ACKNOWLEDGMENT    We thank Dr. Tatuso Ooi for his continued support to the PRF/SEQDB data-base and Dr. Shiro Akabori for inspiring us to undertake this work.                           REFERENCES (1) S. Ohno, "Evolution by gene duplication", Springer-Verlag, 1979. (2) S. Akabori, IUB Symp. I., The Origin of Life on the Earth, I, 189 (1959). (3) M. Go, Nature 291, 90 (1981): Proc. Natl. Acad. Sci. USA, 80, 1964 (1983). (4) Y. Seto, Mol. Design 8, 2 (1987). ( 468 )

Repeat Sequences of Amino Acids Suggest the Origin of Protein (Commemoration Issue Dedicated to Professor Tatsuo Ooi, On the Occasion of His Retirment)

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Repeat Sequences of Amino Acids Suggest the Origin of Protein (Commemoration Issue Dedicated to Professor Tatsuo Ooi, On the Occasion of His Retirment)

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