Correction: A New Interpretation Chicken Transforming Growth Factor-@4 Complementary DNA

of a

David W. Burt and Sonia B. Jakowlew* Department of Cellular and Molecular Biology (D.W.B.) Agricultural and Food Research Council Institute of Animal aid Genetics Research Edinburgh Research Station Roslin, Midlothian EH25 9PS, United Kingdom Laboratory of Chemoprevention National Cancer Institute Bethesda, Maryland 20892

(S.B.J.)

(USB) originally used, revealed six errors (Fig. 1) in the original publication (European Molecular Biology Laboratory Database Accession Number X0801 2): additional nucleotides at positions 77 (C), 359 (C), 364 (C), 365 (G), 391 (G), and a substitution at position 663 (C->A). The revised sequence has a longer open reading frame and is more typical of other TGF-P polypeptides (Fig.

INTRODUCTION The transforming growth factor-p (TGF-P) family is made up of several closely related polypeptides and a number of more distantly related proteins (1). These proteins are capable of influencing the proliferation and differentiation of a range of cell types. Five TGF-P proteins (TGF-Pl to TGF-/35) have been characterized and are all encoded as large precursors. Each polypeptide contains a tetrabasic cleavage site separating pro and mature regions. The degree of amino acid identity between the various TGF-P isoforms ranges from 6482% in the mature and 28-45% in the pro peptides. The individual isoforms are highly conserved throughout the precursor polypeptide. The pro region contains a signal peptide, three Cys residues, several potential Nglycosylation sites and, with the exception of TGF-P2, a RGD sequence (a potential integrin binding site). TGF84 is unique-it lacks a signal peptide and would therefore be a potential intracellular isoform. This conclusion is based on the nucleotide sequence of a chicken TGF04 cDNA (2); however, we now show that the original sequence determination contained six errors. This TGF84 cDNA codes for a typical TGF-6 precursor and, in contrast to the earlier publication, does encode a signal peptide and, therefore, has the potential for being a secreted peptide.

RESULTS

Physiology

2). Amino

Acid Sequence

for TGF-84

The amino acid sequence deduced from the revised TGF-P4 cDNA results in a shift in the reading frame at the 5’-end (Fig. 1). This cDNA is incomplete and lacks a translation start. The amino acid sequence contains a putative signal cleavage sequence, Leu-Ser-Thr-Cys, identical to that found in TGF-Pl (1). Therefore, TGFp4 has the potential to be secreted like any other TGFp polypeptide. A potential tetrabasic recognition site for proteolytic cleavage is found at positions 256-259. Cleavage at this site would release a 114-amino acid peptide. TGF-P4 is unique in this respect, since the other TGF-/3 peptides contain 112 amino acids (1). The mature peptide contains nine Cys residues and, like all other TGF-0 peptides, does not contain any sites for N-glycosylation. The pro region contains three potential N-glycosylation sites, three Cys residues, and a potential integrin recognition motif at position 223. The TGFp4 precursor is larger than that predicted for any other TGF-P protein (371 amino acids and mol wt 41,687).

AND DISCUSSION Sequence

Revision

Deduced

of the TGF-84

Nucleotide

of TGF-/3 Polypeptides

Comparison of TGF-@ polypeptides (Fig. 2) shows greatest sequence conservation in the mature region, including all nine Cys residues. The pro region is less conserved, and only short stretches of sequence identity are found. All pro regions contain a N-glycosylation site at position 82. In addition, both TGF-@1 and TGFp4 have N-glycosylation sites at positions 136 and 176.

Further sequence analysis of the TGF-P4 cDNA using the Taquence system (United States Biochemical Corp., USB, Cleveland, OH) rather than the Sequenase system O&38-8809/92/0989-0992$03.00/0 Molecular Endocrmokqy CopyrIght 0 1992 by The Endcane

Comparison

Sequence

Soaety

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Ala Leu Ser Thr Cy. Gln GC GCG CTC TCC ACG TGC CM Arg CGT 60 ku CTG Trp TGG 120 ~hr KG

Leu Thr CTG XC Lys MG

Glu GM 240 Leu CTG Gly GGG 300 Tyr TAT

Arg Leu Asp Leu CGG CTG GAC CTG 40 Ala Pro Pro Pro Ala Ser Glu Thr GCG CCC CCC CCC GCC TCC GAG KC

Gin Arg Ala CAG CGC KC

Arg CGA

Asp Gly Ala Met Glu GAC GGC GCC ATG GAG Let., La, His Arc, Ala CTG CTG CAC CGC GCC

Gly ~yr GGT TAC 180 Ala Val GCC GTC

Gly Asn &la Ser GGC MT GCC TCG His Gln CAT CM

Trp Leu TGG CTG

Glu Met Arg Ile Ser GAG ATG CGC ATC TCC Ala Met Ala Le" Pro GCC ATG GCG CTG CCG Thr Asp Gl" Lys ACG GAT GAG MG Met Ala Am ATG GCC MC

Am MC

Phe Cya TTC 'SC

Pm Gly Ala Ser Ala CCG GGG &CT TCG GCC

1080 1183

CCCICCMCCCCCCCCCCACMCGAGA~ATTTATTMGTG

Gln Le" Ser As" CAG CTC TCC MT

Met ATG

Glu Ala Ala Lys Lys GAG GCG GCC AM. MG

Ly, AM

Arg Ile Glu Ala Val Arg Gly Gln CGC ATC GAG GCG GTG CGG GGT CM

Ile Le" Ser ATC CTC ,.K

Lys MA

Le" CTG

Pro Pro Arg Pro L-c" Pro Asp Asp Val Arg Ala Leu Tyr Asn Ser Thr Gln Gl" Leu CCC CCC CGC CCC CTG CCC GAC GAC GTC CGG GCT CTG TX MC AGC ACC C&G GAG CTG 80 Le" Arg Pro Pro Pro Asp Gly Pm A.sp Gl" Tyr Trp Ala Lya Gl" Le" Arg A,rg Ile Pro Met Gl" Thr Thr CTG CGG CCC CCC CCG GAT GGA CCC GAT GAG TAT TGG GCC AM GAG CTG CGG CGG ATC CCC ATG GAG ACC ACC 100 Hia Trp Gln Pro Gln Ser Hi3 Ser 110 Phe Phe Vsl Phe Asn Val Ser Arg Ala Arg Arg Gly Gly kg Pro CAT TGG CAG CCG CAG AGC CAC WC ATC TTC TTC GTG TTC MC GTG TCC CGT GCG CGC AGA G&T GGG CG-G CCG 110 Glu Leu Arg "et Leu Arg Gln Lys Ala Ala Ala Asp Ser Ala Gly Thr Gl" Gin Arg Le" Glu LB" Tyr Gin GAG CTG AGG ATG CTG CGG CU MG GCG GCC GCC GAC hGC GCC GGC ACC GM CAG CGG CTG GAG CTG TAC CAG 160 Trp ~cg Tyr Leu His Gly Arg Ser Val hrg Ala Thr Ala Asp Asp Gl" Trp Le" Set Phe Asp Val Thr Asp TGG CGG T,.C CTG CAT GGC CGC TCC GTG CGC KC KG GCC GAC GAT GM TGG CTC TCC TTC GAC GTC ACC GAC 200 Ser Gly Ser Glu Leu Leu Gly Val Phe Lys Le" Ser Val His Cys Pro Cys Gl" Her Gly PIO Gly His Ala WC GGC KC GAG CTC CTG u;G GTC TTC MG CTG AGC GTG CAC TGT CCC TGT GAG ATG GGC CCC GGC CAC GCC 220 ___________ Ile Glu Gly Phe Gl" Gln Gln Arg Gly Asp Met Gln Ser Ile Ala Lys Lys His Arg kg Val Pro Tyr Va.1 ATC GM GGC TTC GAG CW CAG CGC GGG GAC ATG CAG AGC ATT GCC MG MG CAC CGC CGC GTG CCG TAC GTC 260 Ala Gl" Arg Ala Asn Gl" Le" His Ser Ala kg bg lug kg &.sp Le" Asp Thr Asp Tyr Cya Phe Gly Pro GCC GAG CGC GCC MT GAG CTG CAC AGC GCC CGG CGG CW CGC GAC CTC GAC ACC GAC TAC T-SC TTC GGC CCC 280 Gym Cya Val A,rg Pro Le" Tyr Ile Asp Phe Airg Lys Asp Le" Gln Trp Lys Trp Ile His Gl" Pro Lys Gly TGC TN GTG CGG CCG CTC TAC ATC GAC TTC CGC MG GAT CTG CAG TGG MC TGG ATC CAC GM CCC AAA GGT 320 net Gly Pro Cys Pro Tyr Ile Trp Ser Ala Asp Thr Gln Tyr Thr Lys Val Le" Ala Le" Tyr Am Gln His ATG G-GG CCG TGC CCG TX ATC TGG KC GCC GAC KG CAG TAC WC MG GTG CTG GCG CTG TAC MC CM CAC 340 Ala Pro Cy. Cy. Val Pro Gin Thr Le" lrap Pm LB" Pro Ile Ile Tyr Tyr Val Gly Arg Aan Val Arq Val GCT CCG TGC 'KC GTC CCG CAG ACC CTC GAC CCC CTC CCC ATC ATT TAC TAC GTG GGC CGG MT GTG CGC GTG 373 Val Val Arg Ala Cy. Lys Gym Ser GTG GTC CGT GCC TGC MG 'ItiC MC TGA CCCCCCCTCGGG*CCCCCACCACCCCCTTC-C*CC~~CCTCCCCCCCCCCTC

Aan MC 360 Glu GAG

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Fig. 1. Nucleotide and Deduced Amino Acid Sequence of the Revised TGF-P4 cDNA Nucleotides are numbered on the left side and amino acids are numbered on top of glycosylation sites are represented by sing/e overlines. The potential integrin-binding overhne. The 114-amino acid sequence of mature TGF-P4 (boxed-in region) is preceded letters. All Cys residues are highlighted in bold letters. The potential signal cleavage original amino acid sequence (2) is identical to the corrected sequence beyond residue

(Al,,

the nucleotide sequence. Three potential Nsite (Arg-Gly-Asp) is indicated by a broken by four basic residues highlighted in bold site is indicated with a vertical WOW. The 121.

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Chicken

Transforming

Growth

40

30

TGFR5

KFK

TGFR4 TGFDl TGF83 TGFR2 TGFRS

ELLG GEIE ESNL DRNL

Factor+4

991

50

60

EENE

220 GTDE T E L E v Q N G 290 +** TGFl34 NMWRA CS TGFRl NMIVRSC CS TGFR3 NMVVKSC CS TGFl32 NMIVKSC CS TGFRS nil NMVVRSC CS 390

230

240

250

260

270

280

TGFl34 TGFBl TGFl33 TGFD2 TGFR5

300

310

320

330

340

350

360

370

380

Fig. 2. Amino Acid Sequence Similarity Between TGF-@ Precursor Polypeptides TGF-pl (5) TGF-@2 (6) TGF-83 (7) TGF-04, and TGF-05 (8) amino acid sequences were aligned. Residues are numbered relative to TGF-@l Gaps, designated by spaces. are introduced for maximum alignment. Proteolytic cleavage sites are indicated with vertical arrows. Stars represent the Cys residues conserved in all sequences. Potential N-glycosylation sites (NxT or NxS) are underlined. Charged residues are shown (+ or -). The potential integrin-binding site (RGD) is underlined. Amino acid residues conserved (identical and conservative) in all sequences are boxed.

Also conserved in the pro regions of TGF-Pl , TGF-P3, TGF$4, and TGF-P5 is the putative integrin binding site (position 244), which has been detected in several extracellular matrix proteins. TGF-82 is unique in lacking this sequence. All TGF-P polypeptides have Cys residues at positions 33,223, and 225 in the pro region. The longest conserved sequence in the pro region is located at positions 43-59. This region is rich in basic residues and is thought to be required for the association of the pro and mature peptides in the formation of latent TGF-fi (3). The other short stretches of sequence identity in the pro region are important in the assembly, export, stability, and latency of all TGF-@ polypeptides and are discussed in more detail in a recent study (3). The TGF-fi4 sequence is more like TGF-/31 than any other TGF-P protein. The isolation of a chicken TGF-/31 cDNA (4) however, would appear to suggest that TGF84 is encoded by a distinct gene. The TGF-@l and TGFp4 proteins share 82% sequence identity in the mature and 47% in the pro regions, respectively. However, a comparison of any given TGF-/3 isoform isolated from a range of species shows greater than 80% sequence identity in the pro region. This suggests that the TGF81 and TGF-P4 polypeptides are distinct isoforms and so may be secreted and activated differently. The pu-

rification and characterization of TGF-/I4 will be necessary to test this hypothesis. TGF-P4 has only been detected in the chicken, and it remains to be seen if this TGF-P isoform is unique to birds.

Received March 19, 1992. Accepted March 19, 1992. Address requests for reprints to: Dr. David W. Burt, Department of Cellular and Molecular Biology, Agricultural and Food Research Council Institute of Animal Physiology and Genetics Research, Edinburgh Research Station, Roslin, Midlothian Eli25 9PS, United Kingdom. ’ Present Address: National Cancer Institute, Division of Cancer Prevention and Control, Kensington, Maryland 20895.

REFERENCES 1. Roberts AB, Sporn MB 1990 The transforming growth factor-&. In: Sporn MB, Roberts AB (eds) Peptide Growth Factors and Their Receptors, Handbook of Experimental Pharmacology. Springer Verlag, Heidelberg, vol 95:419472 2. Jakowlew SB, Dillard PJ, Sporn MB, Roberts AB 1988 Complementary deoxyribonucleic acid cloning of a mRNA

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encodina transformina arowth factor-B4 from chicken embryo ch&drocytes. MOT Endocrinol 2.1186-l 195 Sha X. Yana L. Gentrv LE 1991 Identification and analvsis of discrete‘iunctionai domains in the pro region of prepro-transforming growth factor beta 1. J Cell Biol 114:827-839 Jakowlew SB, Dillard PJ, Sporn MB, Roberts A 1988 Nucleotide sequence of chicken transforming growth factor-gl (TGF-/31 ). Nucleic Acids Res 16:8730 Derynck R, Jarrett JA, Chen EY, Eaton DH, Bell JR, Assoian RK, Roberts AB, Sporn MB, Goeddel DV 1985 Human transforming growth factor-p complementary DNA sequence and expression in normal and transformed cells. Nature 316:701-705

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Madisen L, Webb NR, Rose TM, Marquardt H, lkeda T, Twardzik D, Seyedin S, Purchio AF 1988 Transforming growth factor-82: cDNA cloning and sequence analysis. DNA Cell Biol 7:1-8 Derynck R, Lindquist PB, Lee A, Wen D, Tamm J, Graycar JL, Rhee L, Mason AJ, Miller DA, Coffey RJ, Moses HL, Chen EY 1988 A new type of transforming growth factor& TGF-P3. EMBO J 713737-3743 Kondaiah P, Sands MJ, Smith JM, Fields A, Roberts AB, Sporn MB, Melton DA 1990 Identification of a novel transforming growth factor-p (TGF-P.5) mRNA in Xenopus laevis. J Biol Chem 2651089-l 093

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Correction: a new interpretation of a chicken transforming growth factor-beta 4 complementary DNA.

Correction: A New Interpretation Chicken Transforming Growth Factor-@4 Complementary DNA of a David W. Burt and Sonia B. Jakowlew* Department of Cel...
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