Conservation of IGFBP structure during evolution: Cloning of chicken insulin-like growth factor binding protein-5

Pergamon

Progress in Growth Factor Research, Vol. 6. Nos. 2-4, pp. 159-165, 1995 Copyright © 1996 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0955--2235/95 $29.00 + .00

PII: S0955-2235(96)00011-7

CONSERVATION OF IGFBP STRUCTURE DURING EVOLUTION: CLONING OF CHICKEN INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-5 Susanne V. Allander,* Ewa Ehrenborg,* Holger Luthman* and David R. Powellt *Department of Molecular Medicine, Karolinska Institute, S-171 76 Stockholm, Sweden tDepartment of Pediatrics, Baylor College of Medicine, Houston, TX 77030, U.S.A.

The insulin-like growth factor binding proteins (IGFBPs) have conserved characteristics of their genomic organization, including similar locations o f exon borders relative to nucleotides encoding conserved cysteine residues. Furthermore, the human IGFBP genes, as well as the human homeobox ( H O X ) genes, are localized to chromosomes 2, 7, 12, and 17. Although little is known about the evolution of the IGFBP genes, the association of human IGFBP and homeobox ( H O X ) genes at four chromosomal loci may indicate that their ancestral genes were linked prior to the first duplication o f chromosomal DNA containing the ancestral H O X cluster. The hypothesis that IGFBPs are ancient proteins is supported by the reported detection of IGFBP activity in serum from the Agnathan species, Geotria australis, a primitive vertebrate. Further studies of IGFBPs in different species are needed to understand the evolution of this protein/gene family. Chicken provides a good intermediate model, since birds diverged from mammals ~300 million years ago. A complementary DNA (cDNA) clone encoding chicken insulin-like growth factor binding protein-5 (clGFBP-5) was isolated. The deduced amino acid sequence is 83% identical to human IGFBP-5 and encodes a mature polypeptide of 251 amino acids. The conservation of IGFBP-5 primary structure across vertebrate species suggests maintenance of important functions during evolution.

Keywords: Gene structure, chromosomal localization, homeobox genes, evolution, insulin-like growth factors, chicken, complementary DNA.

INTRODUCTION Insulin-like g r o w t h f a c t o r b i n d i n g p r o t e i n s ( I G F B P s ) c o n s t i t u t e a s e p a r a t e p r o t e i n family with six identified m e m b e r s [1-3], which b i n d insulin-like g r o w t h

Correspondence to: Dr S. V. Allander. Tel. +46 (8) 729-2096; Fax: +46 (8) 32 77 34. Acknowledgements--This work was supported by Caroline Weiss Law Karolinska-Baylor Research Fellowship, "F6renade Liv" Mutual Group Life Insurance Company, the Swedish Medical Research Council (9109), and National Institute of Health Grant R01-DK38773.

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factors (IGFs) with high affinity and thereby regulate the mitogenic and metabolic activities of IGFs [4]. The six IGFBPs have clearly distinct amino acid sequences, but share regions of homology including cysteine-rich sequences in their N- and Cterminal regions [5]. The diversity within this gene family may have evolved through duplication of an ancestral gene and subsequent transposition to other chromosomes. IGFBP-5 is the most conserved of the mammalian IGFBPs and a short cDNA fragment from Xenopus suggests a high degree of conservation also in nonmammalian vertebrates [6]. The highly conserved nature of IGFBP-5 makes it a good candidate for initial studies of IGFBP structure and function in nonmammalian species. RESULTS AND DISCUSSION Gene Structure and Chromosomal Localization of Human IGFBP Genes

Cosmid clones encompassing the genes for human IGFBP-I to -6 were isolated from a placental cosmid library [7-12]. The authenticity of the cosmid clones were ascertained by further characterization of the genomic organization and DNA sequence of the respective IGFBP gene. The IGFBP genes have their coding parts divided into four exons (Fig. 1), an arrangement now recognized in all members of the human IGFBP family [7, 10-15]. The hlGFBP-3 gene has an additional fifth 3' untranslated exon [14]. The IGFBP genes also have similar locations of exon borders relative to nucleotides encoding conserved cysteine residues [7, 12]. The locations of the 18 conserved cysteine residues relative to exons 1 to 4, is shown in Fig. 1. These cysteines are present in all human IGFBPs, except for IGFBP-6, which is lacking two cysteines in exon 1 [2, 5, 12]. Fluorescence in situ hybridization (FISH) and subsequent quinacrine (Q)-banding localized the hlGFBP-1 to -6 genes to chromosomes 7, 2, 7, 17, 2, and 12, respectively [7-10, 12]. The chromosomal assignments determined by FISH analysis were confirmed by PCR amplifications of somatic cell hybrids with gene specific primers. Table 1 summarizes the reported localizations of hlGFBP-1 to -6. Association Between Human Homeobox (HOX) and IGFBP Genes

The vertebrate HOX genes are suggested to be true homologs to the insect homeotic gene complexes and to have arisen from duplications of a single ancestral gene cluster [19-22]. The human homeobox (HOX) gene family consists of four gene clusters, HOX A-D, localized to chromosomes 2, 7, 12, and 17 [23-27] (Fig. 2). As shown in Fig. 2, the HOX and IGFBP genes/clusters are localized to the same chromosomal regions. Further, the human IGFBP-2/-5 and IGFBP-1/-3 gene pairs are closely linked, with opposite transcriptional orientations [8, 10]. Similarly, the two mouse IGFBP gene pairs (IGFBP-2/-5) and IGFBP-1/-3) are also closely linked [28]. IGFBP-3 and -5 are more closely related to each other than to other IGFBPs, and IGFBP-1 and -2 are more closely related to each other than to IGFBP-3 or -5, which suggests that duplication of an ancient IGFBP gene preceded dispersion of this gene pair to multiple chromosomal loci. Thus, the evolution of the HOX and IGFBP gene families appear to follow a similar pattern. The associ-

Conservation of IGFBP Structure during Evolution

161

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237 aa -25 kb

0.6 kb

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213 aa

FIGURE 1. A schematic IGFBP gene. The filled boxes represent coding parts of exons 1 to 4, and hatched boxes represent 5" and 3' untranslated regions (UTR). I1 to 14 denotes intron 1 to intron 4 and intron lengths are given in kb. The respective IGFBP is indicated at the left and the length of the gene is given within parentheses. The size (in amino acids) of the mature peptide is given at the very right.

TABLE 1.

The localization has been determined by radioactive or fluorescence in situ hybridization (ISH/FISH) and cell hybrid analysis (CH)

IGFBP

Chromosomal localization

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IGFBP-I IGFBP-2 IGFBP-3 IGFBP-4 IGFBP-5 IGFBP-6

7p14-p12 2q33-q34 7p14-p12 17q12-21.1 2q33~134 12q13

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ation of h u m a n I G F B P and h o m e o b o x ( H O X ) genes suggests the presence of their ancestral genes prior to the first genomic duplication o f the ancestral H O X cluster [10]. However, the two m o u s e I G F B P gene pairs do not share the same c h r o m o s o mal localizations as m o u s e H O X A and D [28]. This might reflect two different paths after the divergence of h u m a n s and mice. Therefore, further m a p p i n g o f H O X and I G F B P genes in other, m o r e primitive species, are needed to clarify this point. IGFs and IGFBPs are Ancient Proteins

Existing data suggest that I G F s and I G F B P s evolved prior to the divergence of the primitive vertebrate class A g n a t h a f r o m the m a i n line of vertebrate evolution. It is evident that six separate I G F B P s were f o r m e d before the divergence o f primates f r o m rodents and artiodactyls, a p p r o x i m a t e l y 70 million years ago, since rodent c o u n t e r p a r t s to I G F B P - 1 , to -6 as well as bovine I G F B P - I to -3 have been

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FIGURE 2. Schematic illustration of chromosomal localizations of the human IGFBP and homeobox (HOX) genes. Both gene families have their genes/clusters localized to the following chromosomal regions: 2q31-q34 includes HOX D/hlGFBP-2 and -5, 7plS-p12 includes HOX A/hlGFBP-1 and -3, 12q12-q13 includes HOX C/hlGFBP-6, and 17q12-q22 includes HOX B/hlGFBP-4 [7, 9, 10, 12, 24-27].

identified [1, 2, 29-32]. A cDNA encoding a prepro-IGF molecule has been cloned from hagfish, an agnathan species [33], and IGFBP activity has been detected in agnathan (lamprey) serum [34]. The protochordate amphioxus, which is thought to be the closest living relative of vertebrates, was recently shown to have a single HOX cluster with a genomic organization similar to mammalian HOX clusters [35]. This primitive chordate is known to have a hybrid insulin/IGF peptide [36]. Characterization of Chicken IGFBP-5 cDNA Chicken provides a good intermediate model for studies of IGFBP evolution, since birds diverged from mammals ~300 million years ago [37, 38]. No chicken IGFBP has been cloned prior to this investigation, but multiple IGFBPs have been detected in chicken serum [34], of which one is suggested to be an IGFBP-2-1ike protein [30, 34]. In order to isolate a chicken IGFBP-5 cDNA, probes representing different parts of the human IGFBP-5 cDNA clone 5.12 were designed [10, 39]. A chicken eye cDNA library was screened under stringent conditions and yielded several clones that all contained an insert of ~1.2 kb. The insert from one ~ clone was subcloned and used along with derivated subclones to sequence the complete cDNA clone. The nucleotide sequence contains a 346 bp 5"-noncoding region, an

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FIGURE 3. Comparison of IGFBP-5 amino acid sequences among different vertebrates. The deduced amino acid sequence for chicken IGFBP-5 is compared with the reported sequences for human [39], rat [1], mouse, and Xenopus[6] IGFBP-5, in the region of amino acids 7 to 51.

810 b p o p e n r e a d i n g frame, a n d 80 b p o f 3' u n t r a n s l a t e d sequence. T h e chicken I G F B P - 5 sequence codes for a m a t u r e p e p t i d e o f 251 a m i n o acids, lacking one a m i n o acid as c o m p a r e d with m a m m a l i a n I G F B P - 5 sequences [1, 6, 39]. T h e a m i n o acid substitutions in the chicken sequence, as c o m p a r e d with m a m m a l i a n I G F B P - 5 sequences, are scattered t h r o u g h o u t the c o d i n g region, with p o c k e t s o f highly c o n s e r v e d sequence which m a y suggest i m p o r t a n t functional regions. C o m p a r i s o n to h u m a n , rat, a n d m o u s e a m i n o acid sequences show an overall a m i n o acid similarity o f 8 2 - 8 3 % between chicken a n d either h u m a n , rat, or m o u s e I G F B P - 5 sequences. In a stretch o f a m i n o acid 7 to 51 (Fig. 3) b o t h chicken a n d Xenopus sequences have seven a m i n o - a c i d substitutions c o m p a r e d with the m a m m a l i a n sequences, whereas h u m a n , r a t a n d m o u s e sequences are 100% conserved in this region. W e suggest t h a t the isolated c D N A clone encodes the chicken c o u n t e r p a r t to I G F B P - 5 .

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