Collagen Flashcards
Common features of the Collagen protein family
- composed of 3 separate polypeptide chains (oligomers) called alpha chains
- each collagen can be either a homo- or heterotrimer
- contain regions (domains) of triple helix (quaternary structure)
- primary AA sequence is [gly-X-Y]n
- unusual post-translational modifications of hydroxyproline and hydroylysine
SIDE NOTE post translational mods are also in
elastin, acetylcholinesterase, osteocalcin, and complement component C1q
Physical Forms
1) Fibril-forming: form rope-like structures
2) Network forming: form mesh-like arrays
3) Fibril associated collagen with interrupting triple helices (FACIT): chimeric proteins containing collagen sequences interrupted by sequences homologous to other proteins
4) Transmembrane: link cells with ECM components
Type I Collagen
Physical Form: Fibril-forming
of unique alpha chains: 2 (heterotrimeric)
Major Tissue Distribution: nearly ALL CONNECTIVE TISSUE (skin, tendon, bone, dentin, cornea)
Type II Collagen
Physical Form: Fibril-forming
# of unique alpha chains: 1 (homotrimeric)
Major Tissue Distribution: hyaline cartilage
Type III Collagen
Physical Form: Fibril-forming
of unique alpha chains: 1 (homotrimeric)
Major Tissue Distribution: Distensible connective tissues associated with type I e.g. vascular walls, mucosa and in early wound healing and development
Type V Collagen
Physical Form: Fibril-forming
# of unique alpha chains: 3 (heterotrimeric)
Major Tissue Distribution: nearly ALL TISSUES (esp. placenta, skin)
Type VII Collagen
Physical Form: Network-forming
# of unique alpha chains: 1 (homotrimeric)
Major Tissue Distribution: ANCHORING FIBRILS OF BASEMENT MEMBRANE
Type X Collagen
Physical Form: Network-forming
# of unique alpha chains: 1 (homotrimeric)
Major Tissue Distribution: hyaline cartilage limited to zone of mineral formation
Type IX Collagen
Physical Form: FACIT
# of unique alpha chains: 3 (heterotrimeric)
Major Tissue Distribution: hyaline cartilage in lateral association with type II, proteoglycan NC domains
Type XII Collagen
Physical Form: FACIT
# of unique alpha chains: 1 (homotrimeric)
Major Tissue Distribution: tendons, ligaments, in lateral association with type I, fibronectin NC domains and von Willibrand clotting factor-like domains.
Type XVII Collagen
Physical Form: Transmembrane
# of unique alpha chains: 1 (homotrimeric)
Major Tissue Distribution: epithelial basement membranes (BPAG-2)
**will come across it clinically in an autoimmune disease called bullous pemphigoid which causes the body to make antibodies against collagen XVII
Structural Organization of Type I Collagen
- a heterotrimer made of 3 alpha chains
- extremely tight turns of the alpha helix forces the R-groups of each AA away from the peptide backbone making it PROTEASE RESISTANT! (this is due to steric hinderance)
- short domains at the carboxy and amino termini do not assemble into the collagen triple helix (ICTP and NTx) and are released during collagen degradation
SIDE NOTE “Hole regions” within the collagen fibril allow seeding with hydroxyapatite crystals.
Primary Structure of Type I/ Stability
- composed of repeating [gly-X-Y]n (X = proline and Y = hydroxyproline)
- Glycine at every 3rd position allows for maximal intrahelix hydrogen bonding
- Proline limits rotation about the peptide bonds
- Hydroxproline and hydroxlysine allow for maximal intraCHAIN hydrogen bonding
- Interchain covalent bonds through derivatives of lysin/hydroxlysine form extracellularly (collagen cross-linkages)
- Once denatured by heat and salt, newly synthesized collagen does not reform into native collagen fibrils after cooling but forms a random mass called gelatin.
EM Imaging of Collagen Proteins
Packing of collagen proteins into the growing fibril confers the appearance of periodicity in EM electron micrographs.
Each collagen molecule associates with other collagen molecules in an ordered quarter-staggered array giving the banded periodicity seen in transmission electron micrographs.
Procollagen
- precursor for collagen containing globular carboxy and amino propeptide domains
- Specific carboxy and amino propeptidases synthesized and released by the cell into the ECM at the site of the growing collagen fibril cleave the procollagen amino and carboxy propeptides
Function of Collagen Propeptides
a. Prevention of intracellular fibril formation
b. Registration of the three α-chains by the carboxypropeptides to allow the formation of the collagen triple helix in the cisternae of the endoplasmic reticulum.
Collagen gene transcription
Transcription is “trans-regulated” in that the COL α1(I) and COL α2(I) genes are transcribed in a 2:1 ratio
Regulatory elements exist 5’ and within several introns to coordinate collagen gene expression with nutritional state, functional demands, etc.
hnRNA is spliced, capped, and a polyadenylate tail is added
Then the mRNA is transported to the cytoplasm as a mRNP particle through nuclear pores.
Preprocollagen Translation/Posttranslation
Procollagen mRNA is vectorally translated into cisternae of the ER
The signal peptide is cleaved and α-chains assemble in a C → N direction.
Post-translational modifications (ca. 100/α chain) include:
- Disulfide bond formation.
- Hydroxylation of proline and lysine through prolylhydroxylase (Enzyme requires ascorbate, Fe++, O2, α-ketoglutarate)
- Glycosylation of hydroxylysine
- Extracellular processing of N and C propeptides (Ehlers Danlos)
- Covalent crosslinks between lysine and hydroxylysine derivatives form interchain bonds
Lysyl oxidase requires Cu++ (Menke’s disease) and can be inhibited by ß-amino proprionitrile (Lathyrism).
Osteogenesis Imperfecta
Mutations in type I collagen exons can lead to osteogenesis imperfecta (imperfect collagens cannot pack efficiently into the growing fibril)
The location of mutation within the gene associates with disease severity
Leads to brittle bones
Scurvy
Prolyl hydroxylase requires
ascorbate (vitamin C), Fe++
O2 and α-ketoglutarate for
activity and can only act on procollagen α-chains prior to formation of the triple helix
Under hydroxylation decreases Tm of collagen and the result is scurvy
Why??
Humans can not synthesize
ascorbate due to mutation in
gulonolactone oxidase gene so we need to get it from food and if we don’t get enough…SCURVY YO!
Results in bleeding gums, loose teeth, hemorrhage from intestines
Lathyrism
Aldehyde derivatives of
lysine are essential to the
formation of extracelluar
collagen cross-links
• Lysyl oxidase requires Cu++ as a cofactor. • The enzyme can be inhibited by β-amino proprionitrile resulting in lathyrism
• Menke’s disease: Failure to
extract copper from the diet
Results in lax skin that easily ruptures
Ehler-Danlos Syndrome
Defective extracellular processing of N and C propeptides
Variable phenotype but usualyl lax skin, hyper-extensible joints
Type IV Collagen
Physical Form: Network-forming
# of unique alpha chains: 5 (heterotrimeric)
Major Tissue Distribution:
Major collagen of most basement membranes
Mutations in COL IV exons
also leads to Alport’s syndrome (hereditary nephritis) which causes chronic kidney disease
Organization of the Collagen Gene Family
- Each exon begins with the triplet codon for glycine and each exon ends with the codon for a “Y” amino acid
- For heterotrimeric collagens, genes for the different α chains are always on different chromosomes
- Existence of FACIT collagens. For type XII collagen, portions of non-collagenous protein sequences have been “spliced” from other genes including fibronectin and von Willibrand’s clotting factor.
Functional domain theory of eukaryotic gene organization
Posits that the exon is the nucleic acid equivalent of a functional domain of a protein
So, for collagen, the smallest series of [gly-X-Y] that can form a stable collagen triple helix at 37°C is 6 or 18 amino acids in length, therefore, genetic rearrangements and recombinations of the collagen gene during meiosis would tend to generate new proteins with intact triple helical domains and therefore an increased probability of being a functional protein
General Functions of Non-collagenous Connective Tissue Proteins
- Tissue pliancy
- Volume for tissue growth and cell migration
3 Linking ECM components within a matrix - Cell-matrix interaction
non-collagenous proteinsare what give tissues specificity!!
Elastin
A. Ultrastructure of elastin
1. Amorphous component is 90% of the elastic fiber. 2. Microfibrillar component is 10% of elastin. The percentage tends to decrease with age.
B. Properties of elastin
1. Rubber-like, high molecular weight molecule, seen in all vertebrates. Increased percentage of hydrophobic amino acids is associated with increasing mean blood pressure over phylogeny. 2. Insoluble in most solvents. 3. Amino acid composition: 33% glycine (like collagen) 10-13% proline or hydroxyproline (like collagen) 40% hydrophobic amino acids (DIFFERENT FROM COLLAGEN!!) 4. Contains desmosine: interchain covalent bonds through derivatives of lysine*****
C. Elastin structure
1. Sequence of the elastin gene reveals 2 domains:
a. Small domains that form lysine-derived cross
links.
b. Large domains of hydrophobic amino acids in
random coils.
2. Comparison with collagens (an example of
divergent molecular evolution)
3. Across phylogeny the number of hydrophobic amino acids in elastin correlates with the species mean blood pressure (higher mean blood pressure implies higher metabolic rates).
Fibronectin
A. Properties:
- Abundant in serum and ECM
- Binds collagen, fibrin and other ECM components.
- Facilitates specific cell migration during development and wound healing (eg. neural crest and neutrophil migration).
B. First noted as a protein synthesized by cell cultures that promoted cell attachment, but also found in serum associated with blood clotting proteins.
C. Structure:
- The two fibronectin polypeptide chains are linked via disulfide bonds near the carboxy terminus.
- Fibronectin is composed of 3 types of repeated domains that may have evolved by exon duplication and/or “exon shuffling”. Types of repeats:
a. Type I repeat: also found in plasminogen activator, clotting factor XII.
b. Type II repeat: homologous repeats found in clotting factor XII, prothrombin, plasminogen, urokinase.
c. Type III repeat: found in type XII collagen, contains RGD cell recognition sequence.
D. Cell attachment site.
- RGD (arginine, glycine, aspartic acid), is recognized by some cell adhesion molecules (receptors) called integrins.
- Site may be “displayed” after interaction with other proteins, as in clotting or morphogenesis.
- Fibronectin permits cells that display appropriate integrin cell adhesion molecules to migrate along fibronectin (neural crest migration) or into blood clots (neutrophils) during wound healing.
Collagen Degradation
bacterial versus mammalian
- Fibrillar collagens are highly resistant to most proteinases.
- Bacterial collagenases, such as those expressed by Porphymonas gingivalis, cleave fibrillar collagens at each glycine residue.
- Vertebrate collagenases bind to the hinge region of the collagen triple helix and make a single cut in each α-chain resulting in 1/4 and 3/4 fragments. The resulting fragments denature at body temperature and become substrates for other proteases, such as gelatinases.
- An exception is cathepsin K, synthesized by osteoclasts, that also can degrade fibrillar collagens.
General properties of the MMP family
- In humans, 23 MMPs have been described. At present, all MMPs appear to be products of distinct genetic loci.
- All share a homologous 27 kDa catalytic domain.
- All require Zn2+ at the active site as a co-factor.***
- All require Zn2+ and Ca2+ for conformational stability****
MMAPs are grouped on the basis of substrate specificity into:
a. Collagenases
b. Gelatinases
c. Stromelysins
d. Matrilysins
- Some MMPs are not released extracellularly but remain attached to the cell membrane
Regulation of MMP activity
There are multiple and overlapping mechanisms of regulation exist.
- MMPs are secreted as pro-enzymes locally activated by other proteases such as plasmin or trypsin. Local proteases in turn can be regulated by other proteases such as tissue plasminogen activator, released in sites of inflammation or wound healing.
- MMPs can also be up-regulated by increased transcription/synthesis. Many MMPs are regulated by pro-inflammatory cytokines (IL-1, TNF-α, and others)
MMPS can be INHIBITED locally by tissue inhibitor of metalloproteinases (TIMPs 1-4) and within the circulation by serum α2-macroglobulin.
MMP and Pharmacology
Because Collagen degradation is essential for development, growth, wound healing, tooth movement, etc, MMP expression can sometimes be inappropriately excessive in inflammatory diseases such as rheumatoid arthritis, periodontitis, atherosclerosis, others
In periodontitis, gingival connective tissue degradation appears primarily due to MMP-8***
- Divalent cation chelators such as low dose doxycycline (Periostat®) are clinically used in the management of periodontitis and other inflammatory diseases.
Collagenase 1 (MMP-1)
MMP type: 1`
Source: connective tissue cells
Specificity: Col I, III
Collagenase 2 (MMP-8)
MMP type: 8
Source: inflammatory cells
Specificity: Col I, III
Gelatinase A (MMP-2)
MMP type: 2 Source: connective tissue cells Specificity: gelatin, Col IV-VII, X, XI, elastin and Col I, II, III after 1o cleavage with MMP 1 or 8
Gelatinase B (MMP-9)
MMP type: 9 Source: CT and inflammatory cells Specificity: gelatin, Col IV-VII, X, XI, elastin and Col I, II, III after 1o cleavage with MMP 1 or 8
Cathepsin K
synthesized by mostly by osteoclasts, is a cysteine protease capable of degrading fibrillar collagens but at sites on type I collagen distinct from MMPs 1 and 8.
Cathepsin K as a potential pharmacologic target for future anti-osteoporosis drugs.
