Test 2 lecture 36-37 Flashcards
ECM is made of
- *ground substance:**
- glycoproteins, proteoglycans and glycosamioglycans
- *Fiber components:**
- collagen
- elastin
Collagen breaks into
reticular fibers: lungs, kidneys, lymph nodes
fiber forming:
molecules that help ECM
integrin: bind ECM components together
Metalloproteinases: enzymes that degrade ECM
Growth factors and cytokines:
Transcription factors
to be a collagen:
- must be in the extracellular space and contribute to the structural integrity of the ECM;
- contain at least one triple-helical domain;
- must form supramolecular aggregates (i.e. fibrils (type 1 and 2), filaments (reticular fibers), networks) either alone, or in conjunction with other matrix components.
To be classified as a collagen, the protein must contain at least one ____
triple-helical domain;
wrap around each other and form rope like structure
To be classified as a collagen, the protein must be in the extracellular space and contribute to the ____ of the ECM
structural integrity
To be classified as a collagen, the protein must form ____ (i.e. fibrils, filaments, networks) either alone, or in conjunction with other matrix components.
supramolecular aggregates
type 1 collagen
present in bone, and other tissues (DICT, tendons and ligaments)
is a type of fiber forming collagen
type 2 collagen is found in
cartilage
type of fiber forming collagen
Collagen superfamily: >28 structurally and functionally distinct collagen types with unique ___ exist.
tissue distributions
Some collagen types (e.g. ____) are homotrimers made up of 3 identical polypeptides or alpha “a” chains,
type II collagen in cartilage
Some collagen are heterotrimers made up of combinations of 2 or 3 different a chains (____).
type I collagen in bone
is a heterotrimer of 2 identical alpha 1 chains, and 1 different alpha2 chain
___ form cross-striated linear fibrils and are major structural components of connective tissues
fibrillar collagen
two types of fibrillar collagen
Type 1- bone, skin, tendon, dentin, blood vessels, mesenchyme..
Type 2-cartilage, vitreous of eye
type of collagen that forms non-linear aggregates; have varied function
non fibrillar collagen
examples of non fibrillar collagen
- BASEMENT MEMBRANE: Type IV
- ANCHORING : Type VII
- FACIT : Types IX, XII, XIV
- SHORT CHAIN : Types VIII, X
Formula for triple helical domain:
(GLY-X-Y)n
collagen alpha chains are coiled around each other into a ___ to form a rigid rope-like structure.
triple-helix
___, the smallest amino acid and the only one without a side chain, is critical in every third position of the collagen Gly-X-Y sequence.
Glycine
___ residues occupy the restricted space in the center of the triple helix of collagen
Glycine
Why can only glycine work in center of triple helix of collagen
Any other amino acid in this position disrupts the helix. (would be too big)
In collagen, the side chains in the -X and -Y positions are directed ___, at the surface of the protein, where they participate in intra- and inter-molecular interactions.
outward,
____ have large (~1,000 aa’s per chain) triple helical domains with virtually no interruptions or “imperfections” in the Gly-X-Y repeats.
-Fibrillar collagens
These collagens form straight rope-like structures and laterally associate into quarter-staggered arrays.
Fibrillar collagen
___ have either imperfections and/or large interruptions within the Gly-X-Y repeats.
non fibrillar collagen
These type of collagens are often kinked, and form different supramolecular aggregates.
non fibrillar collagen
Triple-helix structure and stability depends on ___,___,___ and ___ residues.
proline, hydroxyproline, lysine, hydroxylysine
____, are rigid or cyclic amino acids that limit the rotation of the polypeptide backbone, and participate in inter- and intra-chain bonds.
proline, hydroxyproline, lysine, hydroxylysine residues
Supramolecular organization of fibrillar collagen
quarter-stagger array to form fibrils.
hole zones, visible as bands or cross-striations
Further ordering of fibrils depends upon the strength needed in the tissue. (e.g. In bone, type I collagen fibrils are arranged in layers perpendicular to those in the adjacent layers; in cartilage, type II collagen fibrils randomly criss-cross).
Fibrillar collagen molecules align in a ____ to form fibrils.
quarter-stagger array
quarter-stagger array of fibrillar collagen leaves gaps or ___, visible as bands or ____ under an electron microscope.
hole zones
cross-striations
mutations in type 9 collagen causes
early onset OA
early onset OA is caused by mutations in
type 9 collagen
FACITS
How is collagen formed
- Transcription and translation
- Intracellular modifications (cotranslational and post-translational modifications)
- Extra Cellular modifications
type of intracellular modifications when making collagen
- *Cotranslational and posttranslational modifying enzymes:**
1. Removal of signal sequence
- Hydroxylation of some pro and lys residues
- Glycosylation of some lys residues
In the RER or golgi, the α chains fold into trimers:
-Chain association and disulfide bonding in the carboxyl domain
-Trimerization
-Translocation and secretion of procollagen
extracellular modifications when making collagen
Removal of amino (NT) and carboxyl (CT) propeptides (in fibrillar collagens)
Lateral associations into ordered supramolecular aggregates
Intra- and inter-chain lysine-derived cross-link formation
Upon synthesis on membrane-bound ribosomes, each αlpha chain contains a hydrophobic ___ , that is cleaved off the αlpha chains as they enter the rough endoplasmic reticulum (RER).
signal peptide
As αlpha chains, enter the rough endoplasmic reticulum (RER) ____ occurs
trimerization
After trimerization occurs in the RER, the ___ and ___ are cleaved in the ECM
N-terminal propeptide and C-Terminal propeptide
What happens to fibrillar collagen polypeptide when it enters the RER?
Removal of signal sequence
Proline (Pro) and Hydroxyproline (Hypro) represent ___ of the triple helix. These cyclic residues provide ___ by limiting rotation of the polypeptide backbone.
~30%
structural stability
Hydroxyproline is generated by ____ modification, and is essential for ___.
posttranslational
thermal stability
Proline hydroxy groups form ___ which maintain the structure of the triple helix at body temperature.
water-bridged hydrogen bonds
Trimerization of alpha chains does not occur without ____
Hydroxyproline.
Hydroxyproline will ___ the melting temperature of collagen
increase
Tm (melting temperature) of a polypeptide composed of (Pro-Pro-Gly) repeats is
24°C,
while the Tm of a polypeptide composed of (Pro-Hyp-Gly) is 58°C.
___ in the “Y” position are essential for intra-and intermolecular cross-links that stabilize the lateral associations of collagen molecules in fibrils.
Hydroxylysines
Prolyl and lysyl hydroxylases are enzymes in the ER that ___ selected prolines or lysines while the polypeptide chains are growing.
hydroxylate
hydroxylation of selected prolines or lysines occurs before or after trimerization of collagen fibers
before trimerization
___ are enzymes that require ferrous iron and ascorbate cofactors; the ferrous iron is needed at the active site, and is maintained in a reduced state by the reducing agent ascorbate (vitamin C).
Prolyl and lysyl hydroxylases:
Prolyl and lysyl hydroxylases need __ to work.
ferrous iron and ascorbate cofactors
oxygen and aplha-ketoglutarate co-substrates
ascorbate is
vitamin C
needed for Prolyl and lysyl hydroxylases enzymes
ascorbate deficiency
scurvy
The forming collagen aplha chains are underhydroxylated and do not form stable helices at body temperature. Supramolecular aggregates are also destabilized due to reduced cross-linking.
In scurvy, The forming collagen alpha chains are ___ and do not form stable helices at body temperature. Supramolecular aggregates are also destabilized due to reduced ___ .
under hydroxylated
cross-linking
make up of a forming collagen alpha chain
signal peptide
NT(amino terminal) domain-globular domain
TH domain- (Gly-X-Y)n
CT domain- globular domain
-everything except TH domain gets cut off TH will trimerize
Proline hydroxy groups form ___ which maintain the structure of the triple helix at body temperature, ___ melting temperature of alpha helix of collagen
water-bridged hydrogen bonds
increase
cofactors needed for prolyl hydroxylase and lysyl hydroxylase
ascorbate (vit C)
ferrous iron (active site kept in reduced state by vitamin C)
Carboxyl (CT) domains of alpha chains are needed for proper chain alignment prior to ___ formation
triple helix
3 alpha chains are first stabilized by interchain ___ within the CT, followed by a ___ folding of the chains into trimers, starting at the CT domain and proceeding to the amino domain (NT).
disulfide bonds
zipper-like
CT to NT back to front
Most mutations in the CT domain prevent the mutant alpha chain from participating in chain association, and thus cause a reduction in the amount of trimeric molecules formed, leading to ____
“Haploinsufficiency”
Trimeric collagens are translocated in secretory vesicles from the ER or golgi to the ___.
ECM
Removal of amino (NT) and carboxyl (CT) propeptides by N-and C-proteases permits ____ of collagen trimers into fibrils.
lateral alignment
lateral alignment of collagen trimers into fibrils happens after ___ are removed by ___
amino (NT) and carboxyl (CT) propeptides
N-and C-proteases:
Defect is a deficiency in N-protease in the skin, resulting in the persistence of the NT domain, common in cattle and sheep- fragile skin and death
Dermatosporaxis
Dermatosporaxis
autosomal recessive birth defect in cattle and sheep
extremely fragile skin and death
deficiency in N-protease in the skin, resulting in the persistence of the NT domain.
Electron microscopy shows branched, twisted collagens that prevent packing of collagen molecules into cylindrical fibrils.
NT and CT are removed in the ___
ECM,
Once NT and CT are removed in the ECM, collagen molecules align ___ in a quarter-stagger to form___.
laterally
fibrils
Lysines, hydroxylysines and glycosylated lysines are modified by ___ to generate aldehydes that form covalent ___, joining one collagen molecule to the next in the same or another fibril.
lysyl oxidase
cross-links
lysyl oxidase
modify Lysines, hydroxylysines and glycosylated lysines to form aldehydes that form covalent cross-links, joining one collagen molecule to the next in the same or another fibril.
require O2 and copper( in cupric form)
Cross-linking ___collagenous aggregates.
strengthens
Cross-linking defect
lathyrism
Lathyrism.
Cross-linking defects
A. Nutritional: copper deficiency in swine, chicks, sheep leads to blood vessel rupture.
B. Toxic: sweet peas and clover contain β-aminoproprionitrile (BAPN), which binds irreversibly with lysyl oxidase and prevents cross-linking. Collagen is extremely weak, and bones are brittle and misshapen.
defect in CT domain disulfide bond formation
haploinsufficiency
Ascorbate deficiency
scurvy
alpha chains under hydroxylated and can not form stable helices at body temperature-
no hydroxylation= no hydrogen bonds holding triple helix in place= kinks
issues with removal of NT domain from alpha helix
dermatosporaxis
disorder with cross linking of triple helix
lathyrism
prevents cross linking between triple helix, collagen fibril very brittle and weak
BAPN is an inhibitor of
lysyl oxidase
prevents cross linking of collagen to produce fibril
Supramolecular aggregate formations involving FACIT collagens are (collagen Types I & __) and (collagen types II & __).
type 1 (bone) and XII
type 2 (cartilage) and IX
two type of type 1 collagen disorders
osteogenesis imperfecta
Ehlers danlos VII syndrome
osteogenesis imperfecta
brittle bones, incomplete mineralization
Other symptoms may include poor or altered mineralization, osteoporosis, joint laxity, blood vessel rupture, blue sclera.
condition: variable: often lethal in utero, severely deforming or barely noticeable
Lethal: caused by glycine not being every 3rd amino acid in TH region, either deleted or substituted
Collagen type: type 1 (alpha 1 and alpha 2)
ehlers danlos VII syndrome
loose joints and hyperelastic skin
condition: mild to moderate
Type 1 (alpha 1 and alpha 2)
error in the NT domain of collagen alpha helix
Type I collagen
(fibrillar collagen):
- Major protein of the ECM
- Found in bones, teeth, tendons, blood vessels
- Is a heterotrimer composed of two “α1” and one “α2” chains
- Chains associate at the carboxyl domain and fold into triple-helical molecules
- Triple-helical molecules self-assemble into fibrils, which provide tensile strength to the tissue, and serve as templates for mineral deposition.
Based on location and type of mutation, the severity of the disease can be predicted. Mutations in the ___ usually result in less severe phenotypes than mutations in the ____ domain
carboxyl domain
triple-helical
Haploinsufficiency
mutation in the carboxyl domain
this scenario results in a 50% reduction of trimeric collagens produced.
kinked- ends of alpha chains can not line up- trimerization can not occur
Dominant interference
results from the expression of partially-functional collagen α chains that are able to compete with normal chains for binding at the carboxyl domain;
following binding, these hybrid molecules cannot form stable trimers.
lead to either a degradation or abnormal molecules interfere with the normal function
In general, mutations resulting in partially-functional molecules give rise to more severe phenotypes than mutations that prohibit the chains from binding prior to trimerization.
In general, mutations resulting in partially-functional molecules give rise to ___ phenotypes than mutations that prohibit the chains from binding prior to trimerization.
more severe
In haploinsufficiency ___ % of trimers are normal and ___ % are degraded
In dominant interference ___ % of trimers are normal , ___% are abnormal and ___ % are degraded
50% and 50%
12.5 normal, 12.5 abnormal and 75% degraded
Unbranched polysaccharide chains composed of repeating disaccharide units
GLYCOSAMINOGLYCANS / “GAGs
In GAG, 1 of the 2 sugar residues in the repeating disaccharide is an amino sugar (n- ___or ____); the second sugar is a uronic acid (___ or ___)
acetylglucosamine or acetylgalactosamine
glucuronic or iduronic
Gags are usually highly negatively charged due to ___ and ___ on the sugar residues;
carboxyl
sulfate groups
Gags are found in the ___ and on ___
ECM
cell surface
6 gag groups are distinguished by
a) sugar residues
b) type of linkage between these residues
c) number and location of the sulfate groups.
Of these, all but HA are covalently linked to a protein core to form proteoglycans
Of the 6 GAG groups all but __ are covalently linked to a protein core to form proteoglycans
Hyaluronic acid (HA)
IN GAG: ___ bind with glucoronic acid- this is called a ___
and ___ bind with iduronic acid- this is called ___
n- acetylglucosamine (hyaluronan)
n-acetylgalactosamine (dermatan sulfate)
Hyaluronan is a simple GAG, with a long chain of ~25,000 sugar residues and no ___.
sulfate groups
Dermatan sulfate GAG has a high density of negative charges due to ___ and ___.
both carboxyl and sulfate groups
Specific characteristics of each GAG:
Hyaluronic acid (HA):
1) simplest GAG
2) least negatively charged (no sulfate groups)
3) highest molecular weight (>10 6 daltons)
4) not made on a protein core
no sulfate group
5) serves as the backbone for the cartilage PG aggrecan.
Chondroitin sulfate (CS) and keratan sulfate (KS):
1) are highly negatively charged
2) are components of the cartilage PG aggrecan
filled with water, very sponge like- compressive forces
Dermatan sulfate (DS)
1) is a component of two bone PGs, biglycan and decorin
Heparin (HN):
1) most negatively charged GAG
2) found in mast cells
3) has anticoagulant and antiproliferative properties
4) most commonly a component of the PG serglycin.
Heparan sulfate (HS):
1) found on cell surfaces in PGs such as sydecan, glypican, and perlecan
2) proposed to regulate cytokine-mediated cell interactions.
6 GAGs
Hyaluronic acid (HA):
Chondroitin sulfate (CS)
Keratan sulfate (KS):
Dermatan sulfate (DS):
Heparin (HN)
Heparan sulfate (HS):
Linkage between a GAG chain and its core protein in a proteoglycan molecule.
A link tetrasaccharide is first assembled typically on a ___ residue. The rest of the GAG chain, consisting of a repeating disaccharide unit, is then synthesized, with one sugar residue added at a time.
serine
how to make cartilage proteoglycan (aggrecan) biosynthesis
3- protein core of aggrecan.
(4-5) Protein core is transported from RER to golgi.
(6) In golgi, GAG chains are added to the protein core, one sugar at a time.
(7) On completion of glycosylation and sulfation, molecules are transported via secretory vesicles to ECM
(8) . Hyaluronan is made on the plasma membrane
(9) . In the ECM, aggrecan, link protein, and hyaluronan come together to form proteoglycan aggregates
lysyl oxidase is dependent on ___, used to form cross links
Copper
steps to make collagen
- signal protein cut off
- prolyl/lysysl hyrdoxylase and asorbate hydrolyses TH
- CT bind together using disulfide bonds
- TH trimer formed
- secreted into ECM NT and CT cut of (Cross links formed (lysyl oxidase and copper)
4 types of mature proteoglycans
aggrecan (cartilage- very negative, withstand compression (chondroitin sulfate and karatan sulfate))
decorin (smallest, found in bone, make sure distance between type 1 collagen fibrils stays the same(dermatan sulfate))
Serglycrin - made by mast cells, heparin chains
syndecan- cell membrane proteoglycan, heparan sulfate, cytokine metabolism
how to make proteoglycan
- In RER: protein synthesis (make core protein)
- sugars added one at a time to core protein in the golgi
- sugars become sulfated in the golgi
- when fully made, gets packaged into secretory vesicles and moved to ECM
- Hyaluronic acid, made on cell membrane is released into ECM and forms backbone for aggrecan to attach
- link proteins bind these together
