1
Q
Extracellular matrix vs CT
A
ECM is material outside cells and epithelial/endothelial borders
CT is cells AND ECM
2
Q
What tissues are CT?
A
Bone, cartilage, ligament, skin, teeth, tendon, basement membrane, blood vessels, adventitia, cornea, intestinal walls
3
Q
Function of CT
A
- stabilize physical structure of tissues
- ACTIVELY regulate cell behaviour (development, migration, proliferation, cell shape, metabolic functions)
4
Q
Characterization of CT based on chemical composition
A
- proteins
- proteoglycans
- glycoproteins
5
Q
4 steps of elastin fiber synthesis
A
- transcriptions (tropoelastin and microfibril components)
- translation
- secretion
- fiber formation
6
Q
ELN trascription regulation
A
ELN is gene encoding tropoelastin
Increased transcription:
IGF-1/insulin like growth factor 1 and TGF-beta/transforming growth factor beta
Decreased transcription:
TNF-alpha, bFGF (basic fibroblast growth factor), HB-EGF (heparin binding epidermal growth like factor)
7
Q
Tropoelastin
A
- encoded by ELN
- monomer, covalently crosslinked into elastin (making very insoluble)
- secreted by ECM where it associates with microfibrils (fibrillins, MAGPs, fibulins, EMILIN-1)
8
Q
Cross linking of tropoelastin
A
- lysyl residues covalently crosslinked by lysyl oxidase
- makes elastin very insoluble
9
Q
Elastin fiber physical properties
A
expands and contracts (stretches). amorphous
10
Q
Treatment of CT with NaOH and high temperature
A
- solubilizes virtually all proteins
- since elastin is so insoluble, what’s left after treatment is pure elastin
11
Q
elastase
A
cleave insoluble elastin
12
Q
elastin related disorders (6)
A
- cutis laxa
- supravalvular aortic stenosis
- William’s syndrome
- COPD
- Atherosclerosis
- Aortic aneurysm
13
Q
Role of ELN gene in tooth movement
A
- After external pressure, mRNA levels of ELN increased significantly
- ELN gene could then be important in orthodontic tooth movement
14
Q
Marfan’s syndrome
A
-mutation of fibrillin (microfibril related disorder)
Fibrilin mutation –> TGF beta –> matrix metalloproteases –> tissue degredation –>
- Cardiovascular, ocular, skeletal (bone overgrowth and joint laxity) problems
- periodontal disease
15
Q
Proteoglycans
definition, location, physical properties
A
- form highly hydrated, gel like ground substance
- direct and indirect (via other substances being imbedded in proteoglycans) function
- intracellular, membrane associated or extracellular
- carbohydrate rich with some proteins
- bind water and cations
- polyanions
16
Q
Proteoglycans function
A
determine visoelastic properties of joints, withstand compression, permeable, lipid metabolism, hemostasis and thrombosis, matrix assembly and cell adhesion, migration, proliferation, and differentiation.
17
Q
proteoglycan associated disorders (4)
A
- osteoporosis
- osteoarthritis
- Ehler’s Danlos syndrome (progeroid varient, skin fragility)
- corneal diseases
18
Q
Proteoglycans in the oral cavity
A
- postulated roles in tooth development, dentin formation/mineralization, adhesion and fusion of palatal shelves,
- roles remodeling of oral tissues –> involved in periodontal regeneration (decorin up-regulated and biglycan down-regulated)
- perlecan (heparin sulfate) may stimulate bone regeneration bu also may play role in tumorigenesis
19
Q
Proteoglycan structure
A
- core protein
- covalently linked GAG
- proteins and GAG = proteoglycans
- different core proteins, GAG types and number of chains, and chain length of GAG makes differences in PG
20
Q
GAG basic structure
A
- disaccharide repeating unit (long or short)
- have either glucosamine or galactosamine (amino sugar derivatives)
- one neg charged sugar
- neg charged groups either COOH or sulfate groups
- hydration and charge vary
21
Q
Hyaluronic acid
A
heteropolymer of alternating glucuronic acid and N-acetylglucosamine
the only GAG that is not covalently linked to protein core (Free)
22
Q
Major GAGs (6)
A
chondroitin 6-sulfate, keratin sulfate, chondroitin 4 sulfate, heparin sulfate, heparin, dermatan sulfate
23
Q
Aggrecan aggregate
A
- large(!!!), complex, proteoglycan
- aggrecan monomers associate with hylauronic acid to make aggrecan aggregate
24
Q
Glycoprotein subfamilies (3)
A
Fibronectins (widely distributed)
Laminins (basal laminae)
Collagens (most widely distributed protein in body)
25
Glycoprotein-carbohydrate
-glycoproteins covalently bound to carbohydrate:
- the amide NH2 group of asparagine: N linked
- hydroxyl OH group of serine or theonine: O linked
- hydroxyl OH group of hydroxylysine (only in collagens): O linked
26
Role of sugars in glycoprotein
- make more hydrophilic
- help protein folding/tertiary structure
- stabilize against hydrolysis and modulate biding of some components
27
Linker proteins
Some glycoproteins are linker proteins
- Fibronectin
- laminin
- chondronectin: in cartilage; links chrondocytes, type II collagen, cartilage proteoglycans
- thrombospondin: in blood plasma/ECM; links fibronectin, laminin, type V collagen, cell surface receptors, fibrinogen
- vitronectin: bone
- entactin: basal laminae
- nidogen: basal laminae
28
Fibronectin
| location/function
- in ECM/blood plasma
- links types I, III, IV collagen, cell surface receptors, heparin, heparin sulfate, hyaluronic acid
- enable cells to interact with ECM
- determine cell morphology, embryonic cell migration, adhesion, differentiation, wound healing, structural support
29
Fibronectin/cancer
many cancer cells lose ability to synthesize fibronectin: may lead to migration of cancer cells in metastatic cancers
30
Fibronectin in oral cavity
-dental mesenchyme differentiating into odontoblasts regulated by fibronectin
31
Fibronectin structure
- family of glycoproteins
- 5% carbohydrate by weight
- 2 chains linked by disulfide bond near C terminal making V structure
- RGD or RGDS sequence in cell surface receptor binding domain (for binding cell surface receptors and ECM)
32
Laminin
| location/function
- in basal laminae
- links type IV collagen (high affinity), fibrin, heparin sulfate GAGs, other laminin molecules
- enable epithelial cells to interact with ECM
- maintain polarized differentiated phenotype of epithelial cells, embryonic nerve axonal outgrowth, nerve regeneration, adhesion, motility
33
Laminin in oral cavity
- laminin-332 (laminin 5) contributes to growth and terminal differentiation of ameloblasts, and enamel matrix formation
- expression of alpha2 and beta2 chain in fetal oral squamous epithelium and in adult oral squamous cell carcinomas
34
laminin structure
- family of glycoproteins
- 13%carbohydrate by weight
- special domains bind cell surface receptors to ECM
- 3 polypeptide chains (alpha, beta, gamma); disulfide linked heterotrimer
- polypeptides in cruciform (cross) shape
- self associate to form aggregates
35
laminin nomenclature
- indicates chain composition of alpha, beta, gamma chain
| ie. laminin 332 is (alpha3, beta3, gamma2)
36
Integrins function
- transmembrane glycoproteins
- cytoplasmic side binds to cytoskeletal components
- extracellular side binds to fibronectins and laminins (forms basis for linker protein-mediated communication)
- activation via outside in or inside out signalling
37
integrin structure
- transmembrane glycoproteins
- 2 polypeptides (alpha and beta)
- RGD/RGDS sequence on linker proteins binds specificially to integrin receptors
- activation of integrins can lead to clustering and formation of focal adhesions
38
Collagen basics
- structural role (stress bearing), cell attachment, differentiation, chemotaxis
- 1/4 of proteins in vertebrates in collagen by weight
- 28-30 types
39
Steps in collagen fiber formation (7)
- transcription
- translation
- post translational modification
- secretion as procollagen
- processing to tropocollagen (not all, type IV doesn't)
- associate with ECM
- insolubilization into ECM
40
Collagen nomenclature
- 3 polypeptides: either homotrimers (Types II and III) or heterotrimers (Types I, IV, V)
- each polypeptide is alpha chain
- 1 for homotrimers; 1 and 2 or 1,2,3 for heterotrimers
- roman numerals for collagen type designation
ie. Type I collagen: alpha1(I), alpha1(I), alpha2(I)
Type II collagen: alpha1(II), alpha1(II), alpha1(II)
41
Collagen structure
- tertiary: left handed type II trans helix
- quaternary: right handed superhelix
- fibrous proteins
- form insoluble fibers with high tensile strength
- aa composition: 1/3 glycine; charged/positive aa (proline, Hyp/hydroxyproline, hydroxylysine); polar, basic aa also found
- largely repeats of gly-X-Y (often x=pro, y=hyp)--->regular repeating structure
42
Collagen gene transcription control
TGF-beta signal from outside cell to stimulate gene transcription via SMAD
IL-1beta signal from outside cell to stimulate gene transcription
TNF-alpha signals from outside cell to inhibit gene transcription
Several transcription factors interact with enhancer/repressor elements in promoter region
43
collagen gene transcription nomenclature
Eg
| COL1A1, COL1A2, COL3A1
44
Collagen translation
-synthesized on membrane bound ribosomes as larger precursor
45
collagen post translational modifications
- occur cotranslationally
| - include hydroxylation of prolyl and lysyl residues, and glycosylation of hydroxylysyl residues
46
prolyl hydroxylase
- hydroxylates prolyl residues in collagen
- has Fe++ ion at active site
- dioxygenase
- requires ascorbate
- proline hydroxylated at C4 only if pro is on amino side of gly (different enzyme if on carboxyl side)
47
Scurvy
- dietary deficiency of vitamin C (ascorbate)
| - poor calcification of developing teeth (inhibition of collagen formation of dentin & cementum)
48
lysyl hydroxylase
- hydroxylates lysyl residues
- free lysine not a substrate
- hydroxlation of lysine only if on amino side of glysine
- requires ascorbate
49
Ehler's-Danlos Type VI
- lysyl hydroxylase dificiency
| - lowered crosslinking of collagens
50
Collagen glycosylation
- n linked or O linked
- unique to collagens: glycosylated hydroxylysyl residues
- glucose/galactose disaccharide
- degree of glycosylation varies with tissue type
51
procollagen
-after post translational modification two alpha1(I) and one alpha2(I) procollagen associate to form triple helical procollagen
-formed in lumen of ER, packaged and secreted by passing through Golgi
-h bonding between N-H of Gly and O of suceeding reside of other chain in 3 polypeptide chains
-vertically staggered so gly-x-y on same level along helix axis
STRONG
52
Collagen stability
depends on:
- h bonds between N-H of Gly and C=O of second residue in triplet on other chain
- gly as 3rd aa (makes close packing of aa)
- Hyp content (more is more stable, -OH groups also participate in H bonding)
- total imino acid content (more is more stable)
Animals with higher body temperature need higher Tm so more stability.
53
Tm for measuring collagen stability
measure temperature vs viscosity
| Tm is temperature when 1/2 helical structure is lost
54
OI (osteogenesis imperfecta)
- mutation of single glycine to cysteine at residue 988
- triple helix disrupted
- skeletal deformities, brittle bones
55
Ehler's-Danlos Type IV
- defects in type III collagen (gly, skip exon)
- thin, translucent skin
- bruise easily
- rupture arteries
- perforate intestines
- rupture uterus
56
Collagen modification in ECM
- procollagen peptidases cleave N and C terminal of Types I, II, II (not IV) --> product called tropocollagen
- N terminal propeptides have intrachain disulfides, C terminal propeptides have interchain disulfides
- tropocollogen contains entire Gly-x-y repeat region and telopeptides
57
Ehler's-Danlos Type VII
- from deficiency in procollagen peptidases
- hyperextensible skin that bruises easily
- dislocation of major joints
58
Collagen fibril assembly
Types I, II, III
tropocollagen molecules align with 1/4 stagger with 400A gaps between head and tail (site of cross link formation and nucleation site for calcium deposition in bone; also dark part in stains)
adjacent rows have 680A gap between them
59
Cross linking collagen into fiber
- oxidation of lysyl and hydroxylysyl (only in collagen) catalyzed by lysyl oxydases
- spontaneous condensations of products from above form covalent bonds between adjacent tropocollagen molecules
60
lysyl oxidase
- ε amino termini of lysyl residues converted to aldehydes in the presence of O2
- requires Cu++
61
Hydroxypyridinium
AKA pyridinoline
- cross link unique to collagen
- formed by cross link of two hydroxylysyl residues and a lysyl residue in collagen
62
Desmosine
cross link unique to elastin
- 3 lysines converted to allysines via lysine amino oxidase
- aldol condensations form desmosine (or isodesmosine) cross link
63
Collagenases
- cleave collagen
- in family MMP (matrix metalloproteinases)
- cleave in the triple helical region of collagens
- critical to tissue remodeling in vertebrates
- in vertebrates cleave ~1/4 from C terminus
Biochem 3
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