collagen gene structure

8/17/2019 Collagen Gene Structure

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COLLAGEN GENE STRUCTURE

The collagen genes, like most eukaryotic genes, are large, multiexon

nes interrupted at several points by noncoding DNA sequences of unknown function

called introns.54 Thus, the eukaryotic gene coding for a protein is much larger than

would be predicted from the amino acid sequences of the nal protein An example of

the complexity of collagen genes is the intron!exon organi"ation of the type #$$collagen gene ( COL7A I ) %see &ig '()*+, which consists of '' exons, the largest

number in any published gene--

During the early stages of gene expression, the entire gene is transcribed into a high)

molecular)weight precursor m.NA, which is a complementary copy of the coding

strand of the double)helical DNA The precursor m.NA undergoes posttranscriptional

modications, such as capping and polyadenylation, and the introns are removed by

splicing to yield a linear, uninterrupted coding sequence with -/ and */ untranslated

0anking regions The mature m.NA is then transported into the cytoplasm and

translated in cells, such as dermal broblasts

1omplementary and genomic DNA clones corresponding to the α chains of various

collagen molecules have been described in different laboratories These clones have

been extensively characteri"ed and hybridi"ed with the corresponding m.NA

molecules to examine the temporal and topographic expression of these genes by

Northern Not and in situ hybridi"ation techniques, respectively $n addition, their

nucleotide sequence homology with the corresponding amino acid sequences in the

collagen a chains in various animal species has been determined, thus allowing

estimates of the evolutionary conservation of certain segments within the collagens-2

A high degree of conservation implies a region of functional importance within a

protein molecule

.ecombinant DNA technology has also facilitated determination at the precisechromosomal location of the di3erent collagen genes within the human genome %Table

'()4+ 5ith few exceptions, the collagen genes are widely scattered throughout the

human genome &or example, the genes coding for the two constituent polypeptide

chains of type $ collagen, a 1(I) and a4%$+, are located on separate chromosomes, '6

and 6 respectively 7nowledge of the precise chromosomal location of the genes

coding for collagens in human skin will allow development of polymorphic markers

within the genes and in the 0anking DNA for use in genetic linkage studies $n addition,

sophisticated mutation detection strategies, based on scanning of the genes, have led

to identi8cation of a large number of mutations in di3erent collagen genes with

characteristic phenotypic consequences %see, eg, .efs * to -, *', *4, and -6+

TRANSLATION OF COLLAGEN POLYPEPTIDES

9nder physiologic conditions, collagen molecules spontaneously assemble into

insoluble bers This observation presented a logistic problem because it was di:cult

to visuali"e how a collagen molecule could be synthesi"ed inside the cell and then

secreted into the extracellular space without premature assembly of the molecules

into insoluble bers This problem was solved by the demonstration that collagen is

initially synthesi"ed as a larger precursor molecule, procollagen, which is soluble under

physiologic conditions

The precursor polypeptides of procollagen, so)called prepro)a chains, are synthesi"ed

on the ribosomes of the rough endoplasmic reticulum in broblasts and related cells

%&ig '()(+ This initial translation product, the prepro)a chain, contains an amino)

terminal signal %or leader+ sequence The signal sequence, a characteristic feature of


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many secreted proteins, is rich in hydrophobic amino acids and probably serves as a

signal for attachment of the ribosomes to the membranes of the rough endoplasmic

reticulum and vectorial release of the nascent polypeptides into the cisternae of the

rough endoplasmic reticulum During the transmembrane transport of the

polypeptides, the signal sequence is en"ymatically removed in a reaction cataly"ed by

signal peptidase %Table '()*+,The polypeptides released inside the lumen of the rough

endoplasmic reticulum are termed pro- α chains and are larger than collagen a chainsbecause they contain additional peptide sequences at both ends of the molecule

#arious studies show that these noncollagenous extension peptides are di3erent from

the collagenous portion of the molecule in that they do not have glycine in every third

position, they are relatively poor in proline and hydroxyproline, and they are relatively

rich in acidic amino acids These extension peptides also contain cysteine and

tryptophan, which are not present, for example, in type $ and $$ collagens These

noncollagenous domains often contain motifs homologous with sequences found as

building blocks in other extracellular matrix proteins, such as the bronectin $$$

domain, von 5illebrand factor A domain, and thrombospondin N)terminal domain

sequences $t should be noted that, in spite of their homology, these domains do not

have the functional characteristics of the original proteins

POSTTRANSLATIONAL MODIFICATIONS OF POLYPEPTIDE CHAINS

After the assembly of amino acids into prepro)a chains on the ribosomes, the

polypeptides undergo several modications before the completed collagen molecules

are deposited into extracellular bers %&ig '()(+ ;ost of these modication reactions

are cataly"ed by specic en"ymes, and many of the modications are characteristic of

the biosynthesis of collagen %Tables '()* and '()(+ These events are often termed

posttranslational modications to emphasi"e that these reactions are not directly

controlled by the information in the m.NA but occur in the polypeptide chains after

the amino acids have been linked together by peptide bonds The posttranslational

modication reactions of


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6A' A The al %#$$+ polypeptide con8sists of a triple)helical domain that

contains imperfections or interruptions in the ?ly)@) repeat sequence, including a *B

!amino acid ChingeC region The central collagenous domain is 0anked by

noncollagenous segments, the amino)terminal N1)' domain and the carboxyl)terminal

N1)4 domain The N1)' domain consists of submodules with homology to known

adhesive proteins, as indicated below the molecule The N1)4 domain has a segment

of homology with the 7unit" proteinase inhibitor molecule The type #$$ collagen

gene consists of a total of '' exons %vertical blocks+, which are separated from each

other by intervening noncoding intronic sequences %hori"ontal lines+ The si"es %in

base pairs+ of the introns %above

the lines+ and the exons %below

the blocks+ are indicated

%;odied from 1hristiano et

al,- with permission+

A. Intracellular steps

' Translation of prepro)a)

chains on theribosomes of the rough

endoplasmicreticulum

4 1leavage of the signal

sequence* Eydroxylation of selected

prolyl andlysyl residues

( ?lycosylation of some

hydroxylysylresidues

- &ormation of interchain

disulde bonds2 &ormation of triple helices

. Secret!"n "# pr"c"lla$en

C. E%tracellular

&"'!(cat!"ns)

' 1leavage of peptide

extensions by specic

proteases

4 &ibril formation


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* 1ross)linking of collagen brils by deamination of hydroxylysine and lysine

residues to give aldehydes, followed by cross)link formation by reaction of either

%a+ 4 aldehydes or %b+ ' aldehyde and ' F)amino group on adGacent molecules


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TALE *+-

Characteristics of Enzymes Participating in the Biosynthesis of Collagen

ENZYME SUBSTRATE PR!UCT C"ACTRS AN!

CSUBSTRATESysyl hydroxylase

1ollagen galactosyl

transferase

1ollagen glucosyltransferase

Hrotein disulde

isomeraseI

Hrocollagen N)

proteinase

%ADA;Tysyl

oxidases

Nascent prepro)J chains

Hrolyl residue in x)pro)glysequence in pro)a chainsK

Hrolyl resisdue in pro)hyp)

gly sequence in pro)a

chainsK

>ysyl residue in lys)gly, lys)

ser, or

lys)ala sequence in pro)a

chainsK

Eydroxylysine in pro)achainsK

?alactosyl)=)hydroxylysine

in pro)a chainsK

1ysteine residues in the

extensions of pro)a chains

Hrocollagen or pa)collagen

Hrocollagen pc)collagen

>ysyl or hydroxylysyl

residue in brillar collagen

Hro) J chains

()Eydroxyproline

*)Eydroxyproline

Eydroxylysine

?al)=)hydroxylysine

?lc)gal)=)hydroxylysine


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Snt/es!s "# H'r"%ls!ne

Eydroxylysine is another amino acid characteristic of collagen %&ig '()6+ During the

intracellular synthesis of pro)collagen, hydroxylysine serves as an attachment site for

the sugar residues and is critical to the formation of cross)links that stabili"e the

extracellular collagen matrix &ree hydroxylysine is not incorporated into nascent

polypeptide chains, but certain lysyl residues in peptide linkages are converted to

hydroxylysine The hydroxylation reaction is cataly"ed by an en"yme, lysyl

hydroxylase, which, like the prolyl hydroxylases, requires P4, &e4M, a)ketoglutarate, and

ascorbate as cofactors and cosubstrates Despite certain similarities, the prolyl and

lysyl hydroxylases are di3erent en"yme proteins and products of di3erent genes >ysyl

hydroxylase, like prolyl)()hydroxylase, hydroxylates only lysyl residues in the

position of the repeating ?ly)@) sequence Fven though hydroxylation of lysyl residues

in collagen is initiated while the polypeptides are still assembled on the ribosomes, the

formation of hydroxylysine continues for some time after the release of peptides from

the ribosomes

The extent to which lysyl residues in the position of the ?ly)@) sequence are

hydroxylated varies greatly among the collagens from di3erent sources $n particular,type $ and type $$$ collagens are frequently hydroxylated to a lesser degree, so that

these collagens normally contain

approximately four to eight hy)

droxylysine residues per 'PPP amino

acids, whereas type $$ collagen has

approximately four to ve times as

many hydroxylysine residues $n type

$# collagen, most of the lysyl

residues are converted to

hydroxylysine This variation can be

explained in part by di3erences in

the actual number of lysyl residues

that are available for maximal

hydroxylation in the pro)a chains

The variation in the hydroxylation of

lysyl residues can also be explained

by the fact that the nature of the amino acids in the @ position and in the adGacent

triplets in0uences the rate at which lysyl residues in ?ly)@)>ys sequences are

hydroxylated $n addition, lysyl hydroxylase does not hydroxylate a collagen substrate

that is in the triple)helical conformation- Therefore, folding of the pro)a chains into a

triple helix terminates the intracellular formation of hydroxylysyl residues The rate atwhich pro)a chains of di3erent genetic types fold into the triple helix varies, and, in

particular, the rate of triple)helix formation is considerably slower during the synthesis

of type $$ pro)collagen than of type $ procollagen Thus, folding of the procollagen

polypeptides into their triple)helical conformation can regulate the amount of

hydroxylysyl residues in newly synthesi"ed collagen molecules

The critical importance of lysyl hydroxylation of collagen is attested to by the

deciency of lysyl hydroxylase in patients with the scoliotic %type #$+ form of FD


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collagen gene structure

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