Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-05-07
2001-01-09
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069100, C435S320100, C435S325000, C435S252300, C435S254110, C536S023500, C530S356000, C530S353000
Reexamination Certificate
active
06171827
ABSTRACT:
The present invention concerns novel molecules, in particular novel procollagen molecules, together with collagen molecules, fibrils and fibres comprising a non-natural combination of collagen &agr;-chains, DNA encoding same, expression hosts transformed or transfected with same, transgenic animals and methods of producing a non-natural collagen.
Collagen (also known as processed procollagen molecule and triple helical processed procollagen monomeric molecule) (for general reviews see Kadler. K. 1995, Protein Profile, “Extracellular Matrix 1: fibril-forming coliagens”, 2: 491-619, Avad, S. et al., 1994, The Extracellular Matrix Facts Book, Academic Press, London, ISBN 0-12-068910-3 and references therein) is a major structural protein in animals where it occurs in the extracellular matrix (ECM) of connective tissues, mostly in the form of fibrils (also known as polymeric collagen). The collagen fibrils (polymeric collagen) are the major source of mechanical strength of connective tissues, providing a substratum for cell attachment and a scaffold for dynamic molecular interactions. The family of collagens comprises complex multidomain proteins comprising three collagen &agr;-chains wound into a triple helix. At least twenty genetically-distinct collagen types have been described to date and they can be classified into subgroups on the basis of gene homology and function of the encoded protein. Fibril-forming collagens (types I, II, III, V and XI; see Table 1) are synthesized as soluble procollagens (also known as pro&agr; chains, procollagen &agr;-chains and monomer chains) and comprises a C-propeptide, a Gly-X-Y repeat containing region (which in the case of monomer chains of fibril-forming collagens comprise an uninterrupted collagen &agr;-chain) and an N-propeptide. The pro&agr; chains trimerise to form unprocessed procollagen molecules (also known as monomeric procollagen molecules and trimerised pro&agr; chains), assembling into fibrillar structures upon enzymic cleavage of their N- and C-terminal propeptide domains (the N- and C-propeptides) (see FIG.
1
).
Although the genes encoding the pro&agr; chains are remarkably similar, relatively little is known about the processes which control the folding and trimerization of the pro&agr; chains (Dion, A. S. and Myers, J. C., 1987, J. Molec. Biol., 193: 127-143), and only a restricted range of collagens is formed. For example, skin fibroblasts synthesise co-incidentally the six highly homologous pro&agr; chains (pro&agr;1(I), pro&agr;1(III), pro&agr;1(V), pro&agr;2(I), pro&agr;2(V) and pro&agr;3(V)). Despite the great number of possible combinations of the six pro&agr; chains, only specific combinations of collagen chains occur—these are those resulting in types I, III and V collagen. Type I collagen exists as a heterotrimer and assembles with the stoichiometry of two pro&agr;1(I) chains and one pro&agr;2(I) chain ([pro&agr;1(I)]
2
pro&agr;2(I)). Homotrimers of pro&agr;2(I) have not been detected and hence the inclusion of this chain in a trimer is dependent upon its association with pro&agr;1(I) chains. Type III collagens comprise a homotrimer ([pro&agr;1(III)]
3
), and the constituent chains do not assemble with either of the Type I collagen pro&agr; chains. Type V collagen displays heterogeneity with regard to chain composition, forming both homo-([pro&agr;3(V)]
3
) and hetero-trimers ([pro&agr;1(V)]
2
pro&agr;2(V) and [pro&agr;1(V) pro&agr;2(V) pro&agr;3(V)]).
The C-propeptide is known to be implicated in the assembly of the monomer chains into trimerised pro&agr; chains (unprocessed procollagen) prior to cleavage of the N- and C-propeptides and formation of collagen in fibril-forming pro&agr; chains. The assembly of the three monomer chains into trimerised pro&agr; chains is initiated by association of the C-propeptides. This association can be divided into two stages: an initial recognition event between the pro&agr; chains which determines chain selection and then a registration event which leads to correct alignment and folding of the triple helix. Comparison (
FIG. 2
) of the amino acid sequences of the C-propeptides of pro&agr;1(I), pro&agr;2(I) and pro&agr;1(III) pro&agr; chains, which assemble to form collagen types I and III, demonstrates the striking level of sequence similarity between these pro&agr; chains yet, despite the homology, they invariably assemble and fold in a collagen type-specific manner.
It has now been found that the C-propeptides, and more particularly certain sequences within them, are not only necessary but are also sufficient to determine the type-specific assembly of the moieties to which they are attached because of the presence of these certain sequences, the C-propeptides are capable of autonomously directing the assembly of the attached moieties, which in particular may be an alien collagen &agr;-chain. The present inventors have isolated and characterised a region of the C-propeptide which defines the chain selection event but which does not affect the subsequent folding. This has allowed the synthesis of novel pro&agr; chains which have formed novel trimerised pro&agr; chains and collagen. Now that the chain selection interactions between the pro&agr; chains can be controlled, a vast range of novel trimeric molecules, in particular collagens, may be synthesised at will using existing and novel pro&agr; chains and C-propeptides. These new molecules may possess selected biological and physical properties and have a wide range of uses. For example, novel collagens may be used in industries which use collagen either as a product or as part of a process. Such collagens and uses may include for example: novel gelatins for use in food, food processing and photography; novel finings for clearing yeast during the brewing process, novel gelatins for the food packaging industry; novel polymers for the manufacture of textiles; novel glues for use in construction, building and manufacturing; novel coatings for tablets; novel glues for use with the human or animal body: novel collagens for use as body implants; novel collagens and procollagens as adjuvants; novel collagens and procollagens as molecular carriers for drugs and pharmaceuticals; and as modulators of collagen fibril formation for use in, for example, wound healing and fibrosis.
According to the present invention there is provided a molecule comprising at least a first moiety having the activity of a procollagen C-propeptide and a second moiety selected from any one of the group of an alien collagen &agr;-chain and non-collagen materials, the first moiety being attached to the second moiety.
The molecule may be able to bind to other similar molecules. It may trimerise with other similar molecules.
The first moiety will generally be attached to the C-terminal end of the second moiety, although intervening amino acid residues may be present.
The first moiety may comprise a pro&agr; chain C-propeptide or a partially modified form thereof or an analogue thereof, and when forming the C-terminal region of a pro&agr; chain, may allow the molecule to bind to other similar molecules. The C-propeptide region of a pro&agr; chain may be the C-terminal fragment resulting from C-proteinase cleavage of a pro&agr; chain. The C-proteinase may cleave between the residues G and D or A and D or an analogue thereof in the sequence FAPYYGD (residues 376-382 of SEQ ID NO: 2), YYRAD (residues 1-5 of SEQ ID NO: 14) or FYRAD (residues 284-288 of SEQ ID NO: 1) (
FIG. 2
) or an analogue thereof.
Modifications to molecules may include the addition, deletion or substitution of residues. Substitutions may be conservative substitutions. Modified molecules may have substantially the same properties and characteristics as the molecules form which they are derived. Modified molecules may be homologues of the molecules from which they are derived. They may for example have at least 40% homology, for example 50%, 60%, 70%, 80%, 90% or 95% homology.
The present inventors have isolated and identified (see “Experimental” secti
Bulleid Neil
Kadler Karl
Bugaisky Gabriele E.
Carlson Karen Cochrane
Nixon & Vanderhye P.C.
The Victoria University of Manchester
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