Biotechnology in Growth Regulation

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GROWTH HORMONE

SPECIES SPECIFICITY AND STRUCTURE-FUNCTION RELATIONSHIPS OF GROWTH HORMONE

M. Wallis,     Biochemistry Laboratory, School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, U.K.

Publisher Summary

This chapter describes the species specificity and structure–function relationships of growth hormone (GH). GH is a protein hormone derived from the anterior pituitary gland. It comprises a single polypeptide chain of about 190 amino acids, containing two intrachain disulfide bridges. The apparent rapid evolution of GH in the primates makes sequence studies on GH for primates other than man of particular interest. The sequence of GH from the rhesus monkey has been reported. This is very similar to that of human GH, differing at only four residues, suggesting that the rate or evolution of GH because the divergence of old-world monkeys and great apes has been slow. The implication is that GH underwent a period of remarkably rapid evolution early in primate evolution. An alternative explanation is that the primate and nonprimate GH genes are not strictly homologous, implying that a second gene is present in the human genome, homologous with nonprimate GHs and a second gene is present in the nonprimate mammalian genome, homologous with human GH. The situation is complicated by the occurrence of a cluster of GH-like genes in man.

INTRODUCTION

Growth hormone (GH) is a protein hormone derived from the anterior pituitary gland. It comprises a single polypeptide chain of about 190 amino acids, containing two intrachain disulphide bridges. In some species there is a tendency for the polypeptide chain to associate to form dimers or larger aggregates, but it is not clear whether this has any biological significance. GHs are found in all vertebrate groups except possibly the primitive, jawless fishes (Agnatha). There are considerable differences between GHs obtained from different groups, however, in both structure and biological properties.

In this paper I shall survey current knowledge about the species specificity of GHs. concentrating mainly on the differences between the primary structures for which information is available. Some aspects of the relationship between structure and function in GH will also be considered.

MICROHETEROGENEITY OF GROWTH HORMONE

Although for any one species it is normal to refer to GH as a single component, the hormone in many species is known to exist in a number of different forms. As a consequence, GH isolated from anterior pituitaries usually shows microheterogeneity. Such heterogeneity may arise from a number of causes, including the occurrence of more than one gene for the hormone, variant forms of mRNA due to different processing pathways for the mRNA precursors, allelic variation in the population of animals from which the hormone is derived, variable glycosylation (although in most species GH is not glycosylated), limited enzymic cleavage, N-terminal heterogeneity and deamidation. Heterogeneity has been most extensively studied in the case of human GH (20), but occurs in GHs from most other species also. Its physiological significance, if any, is not fully understood, but its possible importance should be borne in mind when species differences and structure-function relationships are discussed.

SPECIES SPECIFICITY OF GROWTH HORMONES: VARIATIONS IN AMINO ACID SEQUENCE

The first GH for which the amino acid sequence was reported was the human hormone (25). Subsequent work on GHs from a number of species produced the sequences of ovine, bovine, horse and pig GHs (summarized in Ref. 40) and revision of the human GH sequence. In 1977 application of the techniques of recombinant DNA allowed the sequence of rat GH to be deduced from the corresponding cDNA/mRNA nucleotide sequence (32). Since then application of both recombinant DNA techniques and conventional protein sequencing has increased the number of complete GH sequences available to at least 20 including 2 primates. 9 non-primate mammals, 2 birds, 1 amphibian and 7 teleost fish. Some of these sequences are summarized in Fig. 1; some are based on protein sequencing, some on cDNA/genomic DNA sequencing and some on both.

Fig. 1 The amino acid sequences of some tetrapod GHs (see opposite)

The availability of sequences for GHs from such a considerable number of species enables detailed comparisons to be made and assessment of possible pathways of molecular evolution in this protein-hormone family. These have been discussed previously (26, 27 41). The additional sequences that have been described in the past few years provide confirmation of the main features recognized previously, particularly with respect to apparently variable rates of evolution.

When the amino acid sequences of non-primate mammalian GHs are compared it is clear that they are very similar. However, when any of these sequences (or a consensus sequence derived from them) is compared with the sequence of human GH the difference is marked (41). The various orders of placental mammals are thought to have diverged at about the same time, 70 million years ago. The divergence time for rat and human is therefore about the same as that for rat and pig; however, rat GH differs from human GH at ∼ 35% of all residues but from pig GH at ∼ 5% of all residues. Such observations suggest that the rate of evolution of GHs in the primates has been much greater than that in non-primate mammals. A variable rate of evolution within a protein family is relatively unusual – normally it is found that the rate of evolution for any individual protein is remarkably constant (though rates vary from one protein to another; eg Ref. 16). The rate of evolution can be quantified by determining a most probable sequence for the GH of the ancestor of the placental mammals (from sequence data available for exisiting mammals) (41). This is very similar to the sequences of pig. fin whale and horse GHs (Fig. 1). The number of amino acid differences observed between the existing GH sequences and this ancestral sequence can then be used to determine the rates of evolution for GH in each of the groups concerned (Table 1).

Table 1

Rates of evolution for growth hormones

*Accepted point mutations/100 residues/10⁸ yr

**’Placental ancestor’ represents the hypothetical GH sequence of the common ancestor of the placental mammals.

A hypothetical (best fit sequence for the GH of the common ancestor of the placental mammals (Anc. p.m.) was derived from available GH sequences, using methods described previously (41). This is shown, using one-letter code. Sequences of various tetrapod GHs are compared with this. A solid line indicates sequence identity; differences are shown using one-letter code; – indicates a gap. Note that the sequences of pig, horse and whale GHs are very similar to the ancestral sequence but those of man, chicken and bullfrog differ from it substantially. Original data for the sequences shown may be found in Refs. 32 (rat), 34 (chicken). 33 (pig). 38 (whale). 29 (bullfrog) or references cited in Ref. 41 (horse, man. ox).

The apparent rapid evolution of GH in the primates makes sequence studies on GH for primates other than man of particular interest. The sequence of GH from the rhesus monkey has been reported (23). This is very similar to that of human GH, differing at only 4 residues, suggesting that the rate of evolution of GH since the divergence of old-world monkeys and great apes (approximately 20 million years ago) has been slow. The implication is that GH underwent a period of remarkably rapid evolution early in primate evolution. An alternative explanation is that the primate and non-primate GH genes are not strictly homologous, implying that a second gene (or family of genes) is present in the human genome, homologous with non-primate GHs (but not expressed as a major form in the pituitary) and a second gene is present in the non-primate mammalian genome, homologous with human GH (but again not expressed in the pituitary). The situation is complicated by the occurrence of a cluster of GH-like genes in man (eg Ref. 12). More information is needed, especially about GH (and GH genes) in other primates. However, it is clear that the nature of GH in primates and non-primates is remarkably different and this must be taken into account when considering the biological actions of the hormone in different species.

The rate of evolution of GHs also appears to have increased, though to a less marked extent, during the evolution of the ruminants (Table 1). The sequences of sheep, ox and goat GHs have all been determined; they show marked similarities. Sequences of any of these ruminant GHs differ from the sequences of rat or pig GHs at about 11% of all residues, whereas the rat, horse, fin whale and pig hormones differ at only 1–5% of all residues.

Most previous discussion of GH evolution has concentrated on mammalian GHs. Sequences of several non-mammalian GHs are now available. In general they are fairly similar to the sequences of the non-primate mammalian GHs. Sequences of GHs from bird (chicken and duck) (5, 34) are more similar to the non-primate GHs than the latter are to human GH, confirming the very rapid evolution seen in the case of primate GHs. GHs from teleost fish show extensive sequence differences from those of mammals, correlating with the lack of activity of these proteins in mammals. On the other hand, partial sequence data available for an elasmobranch fish (21) suggests that here the differences from mammalian GHs are less marked.

Comparison of GH sequences will be influenced markedly by the recent report of the tertiary structure of recombinant pig GH (1). About 55% of the polypeptide chain is in the form of α-helix, folded to give 4 helices in an antiparallel, twisted helical bundle. Sequence comparisons indicate that the α-helical regions are rather more conserved than other parts of the molecule, suggesting that a similar conformation is found in GHs from other species, and possibly in other proteins of the GH family.

On the basis of sequence comparisons, GHs are thought to be members of a family of homologous proteins along with prolactin and placental lactogens. Comparison of sequences of bovine GH and prolactin (eg Ref. 40) shows identity at about 25% of all residues, enough to establish homology but less similarity than is seen within the GHs (though teleost and mammalian GHs showquite low homology; eg 32% identity for GHs from man and tuna – Ref. 30). Distinct GHs and prolactins are known to occur in most fish groups and it seems likely that GH and prolactin diverged following a gene duplication early in vertebrate evolution.

The relationship of GHs to placental lactogens is more complex. The best-characterized placental lactogen is the human protein. This has an amino acid sequence very similar to that of human GH. differing at only about 15% of all residues. Indeed, human GH resembles human placental lactogen much more closely than it does any of the non-primate GHs. This implies that human placental lactogen diverged from human growth hormone (presumably following a gene duplication) during the evolution of the primates, after their separation from the other main orders of placental mammals (41). In accordance with this it is notable that the genes for placental lactogens lie very close to those for GHs in man (12). The situation in non-primates is different. Many non-primates possess placental lactogens. Such proteins have been less fully characterized than the human placental lactogen, but in the case of the rat and mouse, for which sequences have been reported (7, 15). it is clear that the placental lactogens are more closely related to the prolactins than they are to GHs. A similar situation probably occurs in ruminants (31). In the rat and mouse there are several different forms of placental lactogen-like proteins, together with another prolactin-like protein, proliferin, first identified as expressed in proliferating fibroblasts, but now identified as produced also in the placenta (17). The physiological roles of these various placental proteins are still to be elucidated.

SPECIES SPECIFICITY OF GROWTH HORMONES: VARIATIONS IN BIOLOGICAL PROPERTIES

Species specificity was observed early in the isolation of GHs, when it was recognized that non-primate mammalian GHs had little anabolic activity in man. GH from humans is active in man (and in most other species tested) and this has formed the basis of the widely-used treatment for hypopituitary dwarfism. In accordance with their low growth-promoting activity in man, GHs from non-primates also show only low ability to bind to GH receptors derived from human cells or tissues (eg Ref. 18). These differences between the biological activities of human and non-primate GHs presumably relate to the marked structural differences between the proteins, discussed above, although the precise differences underlying them are not clear. It is notable also that human GH shows much more marked lactogenic properties, in a number of systems, than the non-primate GHs.

GHs from teleost fish appear to have little growth-promoting activity in mammals, though mammalian GHs are active in fish. Again the large differences between the amino acid sequences of GHs from teleost fish and mammals presumably underlie the differences in activity.

GHs from different species vary considerably in their ability to bind to ‘receptors’ prepared from different tissues. Receptors in membrane preparations prepared from rabbit liver have been most studied (4, 14, 39, 47). In this system rat, mouse and pig GHs appear to be much less active than the human, bovine or ovine hormones (4, 47). These various hormones all have very similar growth-promoting potencies when assayed in rats. It is not clear what underlies their different abilities to bind to rabbit liver receptors, but the results do suggest that potencies in radioreceptor assays do not correlate well with those determined by bioassay. The activities of GHs and prolactins from various species in radioreceptor assays from a number of tissues and species have been surveyed and discussed in detail by Nicoll et al. (28).

STRUCTURE-FUNCTION RELATIONSHIPS

A good deal of work has been reported on the investigation of structure-function relationships in GH and related proteins, based on comparison of sequences from different species and on chemical and enzymic modification studies. The recent description of the tertiary structure of pig GH will clearly influence future consideration of such relationships, and the production of variants using recombinant DNA techniques should provide a new source of well-defined derivatives. Only a few of the results obtained on structure-function relationships can be considered here. A detailed and thoughtful discussion of structure-function information that can be derived from sequence comparisons has been presented recently by Nicoll et al. (27).

Disulphide bridges

All GHs described so far have two disulphide bridges, one linking distant parts of the primary structure (in bovine GH residues Cys-53 and Cys-164). the other forming a loop near the C-terminus. The C-terminal disulphide bridge is relatively easily modified; selective modification leads to very little loss of growth-promoting activity (10, 11). In human GH reduction of both disulphide bridges can be achieved without using denaturing conditions. If the cysteine residues so produced are modified by alkylation with iodoacetic acid (with introduction of additional negative charges) the hormone loses biological activity, but alkylation with iodoacetamide gives a form of the hormone in which biological activity, and tertiary structure, is retained (6, 11). The activity of human GH in which the disulphide-bridge structure is disrupted has also been demonstrated by production of an active derivative in which Cys-165 is substituted by alanine (37) by recombinant DNA methods. Thus the tertiary structure and biological activity of the molecule is not dependent upon intact disulphide bridges, though it cannot be ruled out that such bridges stabilize the molecule in vivo.

Limited enzymic cleavage

With a number of specific enzymes. GHs can be cleaved very selectively to give discrete derivatives. Cleavage of human GH with plasmin provides a much-studied case. The enzyme cleaves after only two residues, removing a hexapeptide from the molecule and leaving a two-chain GH in which the two chains are held together by a disulphide bridge. The tertiary structure of the protein appears to be retained in the derivative, and plasmin-digested human GH appears to retain full biological activity (24); indeed, there have been some suggestions that such plasmin treatment can lead to GHs with enhanced activity, though this is controversial. After reduction and alkylation of disulphide bridges the two chains can be separated. The larger. N-terminal fragment retains significant, low biological activity. Full activity can be regained by allowing refolding of the two chains under appropriate conditions, and this has allowed some studies on the importance of the C-terminal fragment for biological activity (22).

The ability of plasmin-derived fragments of human GH to interact with antibodies has also been investigated (43). A panel of 7 monoclonal antibodies was unable to distinguish between intact GH and plasmin-cleaved GH. indicating that the residues removed by plasmin (residues 135–140) are not involved in antigenic determinants recognized by any of these antibodies. When the N- and C-terminal fragments of plasmin-digested GH were separated, ability to interact with antibodies was largely lost in the case of most of the monoclonal antibodies and a polyclonal antibody to human GH. but two of the monoclonal antibodies bound effectively to the N-terminal fragment, indicating that the epitope to which they were directed is retained in this fragment; it is presumably sequence-dependent or directed against a component of the tertiary structure that is retained in hGR1–134.

Plasmin digestion of bovine GH is less specific, and gives rise to a less well-defined mixture of derivatives than is seen in the case of human GH (35).

The multiple actions of growth hormone

GH is known to have a range of actions (42). In addition to (and perhaps components of) its actions on growth promotion, it stimulates production of somatomedin C/IGF-I and a number of other proteins, and has a variety of metabolic effects including insulin-like and diabetogenic actions on carbohydrate metabolism, and lipid mobilizing effects. It has been a matter of debate for many years as to whether all of these actions stem from a single primary event (presumably interaction of GH with a single receptor) or whether they involve interaction with a number of different receptors, recognizing different regions of the tertiary structure of the hormone.

Evidence for multiple receptors for GH has come from a number of approaches, including studies using monoclonal antibodies to the hormone (36). monoclonal antibodies to the receptor (3). binding specificity (4, 14, 47) and crosslinking reagents (44, 50). However, it remains unclear whether the different receptor types identified can be associated with specific actions of the hormone. The recent cloning of the GH receptor (19) has demonstrated that at least some of the multiple receptor types are probably products of a single