J.-L. Vilotte
a-lactalbumin (alac), a major whey protein, is a calcium metalloprotein (123 aa in most species, 122 in rabbit and 140 in rat), that has been found in all milks studied so far. It interacts with UDP-galactosyltransferase to form the lactose synthetase (1 for review) and was thus described as a key protein for lactogenesis (2). Other functional properties have been attributed to this protein such as a cell lytic activity (3), induction of cell growth inhibition (4) or apoptosis (5).
The amino acid sequence of the alac preprotein has been determined either directly or deduced from its cDNA in at least 14 species (review in 6, 7). It revealed the occurence of a 19 aa signal peptide (20 aa in mice), one of the most divergent among those of the major milk preproteins, and significant sequence homology with lysozyme (8 for recent review), as does the protein three dimensional structure (9). It is hypothesized that the two protein-encoding genes share a common ancestor, duplication of which leads to the appearance of alac and lactation. Occurrence in Echidna of a lysozyme with alac activity reinforced the evolutionary potential relationship between the two proteins (10). alac sequence comparison has also highlighted the conservation of amino acids to which functional roles were attributed (11). Recently, studies on mutated alac produced in various systems such as E.Coli (12) or baculovirus-infected insect cells (13) have confirmed the role of some of them.
The early hypothesized common ancestral origin of alac and lysozyme was also confirmed by the similar structural organization of the relevant transcription units (14 and Fig.1), but also by the induction of enzymatic activity to alac by shuffling exon 2 of the gene with that of lysozyme (15). In this review, we will attempt to summarize our knowledge on alac gene structure and expression and on its use in transgenic experiments.
Structural analysis of alac-encoding genes and related sequences
The alac-encoding transcription unit has been characterized in seven species (16 for
review and 17). All but the Tammar Wallaby (17) genes share a short transcription unit (of
around 2kpb) split into four exons (Fig. 1). Their 5' flanking sequences are quite well
conserved, with a putative TATA box and other potential trancription factor binding sites
(16, 18). Four exons were also identified in the Tammar Wallaby alac gene transcription
unit, but occurrence of a fith one, 5' to the four sequenced, was hypothesized due to the
lack of putative regulatory sequences within that region (17). It remains however to be
fully established. Human, bovine, goat and sheep alac genes were assigned to chromosome
12, 5, 5 and 3, respectively (19-21).
Occurrence of alac related sequences was observed by Southern analysis in the bovine (22) and goat (23) species and some of them were characterized in the bovine and ovine species (22, 24). All contain a nucleotide stretch sharing high homology (over 78 %) with the functional gene downstream of exon 2 that is flanked by repetitive DNA, such as LINE sequences (Fig.1). So far, such an alac multigene family was only described in ruminants. Similarly a lysozyme multigene family was found in ruminants (25-26). These two mutigene families share the occurrence of LINE elements, potentially implicated in the evolutionary process at the origin of their appearance (27), and in the bovine genome alac and all but one lysozyme sequences are located on the same chromosome (28), suggesting a potential concerted evolution. However, alac sequences were found to be outside the lysozyme gene cluster (28).
Endogenous alac gene expression and regulation
Despite various reports, expression of alac appears to be restricted to the mammary
epithelial cells. RNA and protein levels are low in virgin glands and during most of
pregnancy, arise sharply near parturition and decrease rapidly following involution (16,
29-30). This pattern of expression differs from that reported for other major milk
proteins (31). Heterogeneous expression of alac and other milk proteins genes in ruminant
mammary tissue was reported (32). Hormonal regulation of alac gene expression or protein
synthesis has been mainly studied using mammary explants derived from mid-pregnant rat or
mice (33 for review). In contrast with casein or WAP genes, cortisol, in the presence of
insulin and prolactin, has a dose-dependent action on alac: it activates the gene
expression at low concentration (30 nM) but inhibits it at concentrations above 300 nM
(34-36). Prostaglandins reversed this inhibitory effect of cortisol (37). Furthermore,
again in contrast with casein genes, insulin and cortisol alone or with thyroid hormones
can stimulate alac expression (38-39). Prolactin in the presence of insulin and cortisol
can slightly enhanced alac mRNA accumulation within 4 to 6 hours of culture and alac
protein only after 24 hours of culture (40). Cyclic AMP and progestin suppress alac
expression (41-42) but apparently only in presence of prolactin for progestin (43).
Explants derived from lactating glands did not respond anymore to progestin and cortisol
(29,44). Marsupials' alac gene regulation appears to be more dependent on prolactin than
in eutherians (45).
Expression of alac transgenes
Bovine, goat and guinea-pig alac genes have been expressed in transgenic mice (46-48) and
rats for the bovine gene (49). Overall, expression of the gene was found to be
tissue-specific and developmentally regulated, and level of the recombinant protein in
milk reached 3 to 4 mg/ml. However, expression of transgenes with 5' and 3' flanking
sequences as long as 8.5 kbp and 9.5 kbp, respectively, was dependent from the integration
site and unrelated to the number of copies integrated, suggesting the lack of
cis-regulatory sequences such as insulators. Comparative expression analysis of
5'-shortened constructs revealed that the region -477/-220 contains important cis-acting
transcriptional elements (47). A potential STAT5-binding site was identified within this
region (16).
The 5' flanking region of bovine alac gene has been used to direct secretion of foreign proteins into the milk of transgenic mice (50-52) or rat (53). Low frequency, low level but apparent tissue-specific expression of an alac/ovine trophoblast interferon transgene was observed in mice (50). This poor result probably reflects the use of an intronless construct. The alac promoter was able to efficiently target expression of bovine b-casein (51-52) and human growth hormone (53) hybrid genes, resulting in the occurrence of several mg of recombinant protein/ ml of milk (up to 10 mg/ml, 51). Low levels of human growth hormone were detected in the serum of rats from highly expressing trangenic lines, a leakage also detected using other milk protein gene promoters (53). In mice secreting high amounts of bovine b-casein, a surprising abrupt involution occurred that appeared correlated with the level of recombinant protein detected in the milk (51). However, this observation was not reproduced in a recent experiment involving expression of mutated b-casein, despite similar levels of expression (52). Altogether, these results suggest that the alac promoter can be an efficient vector to direct expression of hybrid genes to the lactating mammary gland.
Alteration of endogenous alac expression in transgenics
Lowering the lactose content of milk is an important goal that could result in the
production of a more concentrated milk and/or a better nutritional source for lactose
intolerant people (more than 90% of the adult population) and industrial processing. A
direct approach, currently being tested, is the synthesis of an active lactase in the
milk. Alternatively, alteration of alac expression could result in a lower milk lactose
content, provided alac is solely responsible for lactose synthesis in vivo and has no
other key biological role. Recent obtention of alac-deficient mice using ES cells and
homologous recombination confirmed both assumptions (54-55). Such mice produced a highly
viscous milk, devoid of lactose, with higher protein and fat contents, that pups were
unable to suck out. No other related phenotype was observed. It thus demonstrated the
feasibility of altering milk composition by lowering the alac gene expression.
Double gene replacement in ES cells has been used to substitute the human alac gene to its endogenous counterpart in mice (55-56). This experiment resulted in the high expression of the human gene and the protein was shown to induce lactose synthesis in murine alac-deficient mice (54), as did bovine alac also (57). This technology can be applied to introduce subtle mutation in the gene to lower its expression. However, true ES cells are yet to be isolated in large animals, limiting so far this approach to mice. Recently, expression of ribozyme targeted against the bovine alac mRNA was shown to significantly (up to 50%) and specifically reduce levels of these transcripts in double transgenic mice that normally secreted 0.4 mg of the recombinant protein per ml of milk (58). This strategy could be used in large animals but will require first the development of more efficient transgenes in terms of frequency of expression.
alac plays a key role in lactogenesis. This protein presents much interest in various fields such as the structure/function relationship of both the protein and the gene, evolution and more applied research on alteration of milk composition (lowering lactose or production of foreign proteins in milk). Surprisingly, compared to some other major milk protein genes, little is known on the location of cis-regulatory elements within this gene.
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Keywords:
contributed by Jean-Luc Vilotte
INRA-CRJ
78352 Jouy-en-Josas
Cedex, France
Fax: (33 1) 34 65 24 78
E-mail: vilotte@biotec.jouy.inra.fr
(e-mail vilotte@biotec.jouy.inra.fr)
Dr. Hubert Leveziel
Laboratoire de Genetique biochimique
et de Cytogenetique
INRA, CRJ
78350 Jouy-en-Josas
FRANCE
Tel : 33 1 34 65 25 80
Fax : 33 1 34 65 24 78
Mail: leveziel@biotec.jouy.inra.fr
contributed: September, 1996 |
