ECM 1 and breakout

Define Glycosaminoglycan

(GAG)- The carbohydrate portion of proteoglycans. -GAGs may contain up to 100 sugars per chain with specific repeating sequences composed of a uronic acid and an amino sugar. These carbohydrates are polyanionic due to uronic acid and high level of sulfation on the amino sugars

Characteristics of Keratan sulfate

Has three different subclasses Links to core protein using glycoprotein structures

Dermatan Sulfate

Higher charge density than other proteoglycans Structurally similar to chondroitin sulfate Disruption of dermatan sulfate synthesis can lead to many mucopolysaccharidosis Maroteaux-Lamy syndrome or polydystrophic dwarfism which is a defect of synthesis of DS Hurler's syndrome which is a defect of catabolism of DS

Describe the multi-step process of Collagen synthesis and secretion...

1. Preprocollagen is synthesized containing a signal sequence directing it to the lumen of the endoplasmic reticulum (ER) 2. Removal of this signal sequence results in the formation of procollagen 3. Post-translational modification occurs in the ER Hydroxylases convert proline to hydroxyproline Glycosylation attaches sugars to the protein Disulfide linkages are incorporated 4. The disulfide linkages trigger formation of the triple helical structure producing tropocollagen 5. Tropocollagen triple helices are transported to the Golgi apparatus and then exocytosed 6. Tropocollagen is then converted to mature collagen by proteolysis 7. Mature collagen can then be crosslinked to form the insoluble collagen fibrils

Extracellular Matrix (ECM) functions:

1. Provides shape and structure to the interstitial space 2. Provides lubrication and cushioning to cells and other biological products (e.g. inorganic bone structure) 3. Provides an anchor point to allow cell adhesion 4. To some extent allows communication between adjacent cells and controls cell life cycles Signaling of this type can control proliferation, differentiation, migration and apoptosis among other things

This highly charged matrix leads to three important properties of the ECM. What are they and why are they important?

1. Rigidity 2. Flexibility 3. Compressibility This overall structure allows tissues to withstand torsion and shock This is especially important in the biomechanical tissues of joints, muscle and bone

Major proteoglycans of the ECM include the following:

Hyaluronic acid Chondroitin sulfate Dermatan sulfate Heparan sulfate Keratan sulfate

Loss of chondroitin sulfate in joints results in...

Loss of chondroitin sulfate in joints is a cause of osteoarthritis

What is the main function of fibronectin? To what will fibronectin bind?

Fibronectin is an ECM that acts as an attachment point. Binds to pretty much everything in the ECM. It is involved in Cell Adhesion, Cell migration, Embryonic Morphongenesis, and cytoskeletal and ECM organization. Fibronectin is tissue specific in it's binding mechanisms. Typically found in the basement membrane of cells.

Hyaluronic Acid

AKA hyaluronan or hyaluronate The polysaccharide chain is the longest of the GAGs No protein core No sulfation Major component of the synovial fluid and vitreous humor. (Increases viscosity of the fluid and is one of the fluid's major lubricating components) Also present in ECM of cartilage and the skin Involved in inflammation and wound repair UVB sunburns result in decreased production and increased breakdown of hyaluronic acid leading to inflammation and erythema

Nonfibrillar collagens include many different types:

Basement membrane collagen (Type IV) Fibril-associated collagens with interrupted triple helices (FACITs) Collagens with multiple triple helical domains with interruptions, called multiplexins

Where is Gly, Proline and hydroxyproline oriented in Collagen?

Gly residues are oriented toward the center of the triple helix because of their very small side chain Proline and hydroxyproline residues provide strength to the collagen structure due to their bulk and rigidity

Collagen

Collagens are the major protein components of the ECM. (They are also a major component of the body comprising about 30% of total protein mass) More than 25 different collagens have been identified to date Each of these collagens are related proteins but differ in their distribution, organization and function in tissues

Osteogenesis imperfecta (Brittle bone disease)

Congenital disease caused by defects in the synthesis of Type I collagen Usually genetic defects in the genes coding for α1(I) or α2(I) collagen chains Usually a point mutation in the coding of the Gly-X-Y repeat leading to bulky amino acids in the glycine position Changed amino acid sequence prevents formation of the triple helical structure Characterized by fragile bones, thin skin, abnormal teeth and weak tendons

mucopolysaccharidosis

Disruption of either of these processes (construction or degradation of proteoglycans) leads to a broad class of diseases called mucopolysaccharidosis

Describe the specific structure of collagen...

Each collagen protein has a unique left-handed helix (this is different from a standard α-helix seen in other proteins) There are three amino acids per turn of the protein (an α-helix has 3.6 per turn) Each turn of the helix has a specific sequence with the sequence Gly-X-Y where X is predominantly proline and Y is predominantly hydroxyproline Three helical collagen proteins come together to make a triple-stranded right-handed superhelical quaternary structure

Describe how desmosine forms in the ECM. Be specific as to its precursors

Elastin is a protein that is formed that has few post-translational modifications. In the extracellular space, elastin monomers are crosslinked to form a complex two dimensional latticework, producing a structure called desmosine. nitrogen comes from Lysine in the structure. we get a condensation rxn forming a pyramidine (spelling?) ring.

What are the two classes Collagen types can be broadly categorized into?

Fibrillar (fibril-forming) collagens Nonfibrillar collagens

What is Fibronectin?

Fibronectin is an ECM protein that acts as an attachment point for other cellular and ECM components At least 20 different isoforms of fibronectin have been identified and each has its own binding specificity Isoform production has been shown to be both tissue specific and developmental stage specific Involved in cell adhesion, cell migration, embryonic morphogenesis, and cytoskeletal and ECM organization

Characteristics of Hyaluronic acid

Has no core protein Has no sulfation Major component of synovial fluid

What is desmosine?

In the extracellular space, elastin monomers are crosslinked to form a complex two dimensional latticework Because of the structure of these latticeworks, elastin polymers can stretch in two dimensions

KS-II

KS-II is found primarily skeletal tissue including bone and cartilage Linked to serine or threonine via N-acetylgalactosamine

KS-III

KS-III is found primarily in the brain Linked to serine or threonine through mannose

Define Aggrecans

Large macromolecular bottlebrush-like structures that many different proteoglycans interact to produce. Hyaluronic acid is the core protein.

Characteristics of Heparan Sulfate

Most charged GAG Related to a common anticoagulant

What is Chondroitin Sulfate and whats its function?

Most common GAG Major component of cartilage Function is to resist compression of cartilage through electrical charge repulsion of the negatively charged sulfates

Characteristics of Chondroitin Sulfate

Most common GAG Major component of bone and cartilage

Keratan Sulfate

Most heterogeneous GAG Three classes of keratan sulfate which differ in the amino acid of protein attachment

Heparan Sulfate

Most highly charged of all proteoglycans Major component of the basement membranes Binds many soluble regulatory proteins including interferon-γ, growth factors and antithrombin III Heparin is a small structurally related soluble GAG which has strong anticoagulant properties Binds to antithrombin II and inhibits the clotting cascade

What is the etiology of the mucopolysaccharidosis? What is the etiology of the lysosomal storage diseases?

Mucopolysaccharidosis is a broad class of diseases caused by a disruption of one of two processes. Can't synthesize or degrade because of defective enzymes. The first process that may be disrupted is the construction of proteoglycans constructed in the Golgi App, the second is the degradation of proteoglycans in the lysosomes. If break this sequence then it won't work. the breakdown is stored in the lysosome.

Why might the basement membrane act as a barrier to cancer development? How might cancer cells target the basement membrane to grow and spread?

The basement membrane acts as a barrier (mesh like structure) preventing certain cells from crossing over into other tissues and organs. Cancer is uncontrolled growth of cells so the basement membrane prevents these cells from spilling over into areas and tissues were they shouldn't be. Cancer cells can be mutated to break down the BM and allow cells to metastasize. Type IV collagen is the major collagen type in the basement membrane so any cell secretions (enzymes secreted by cancer cells) that break down collagen. then the structural support would weaken the basement membrane and would allow it to be broken or penetrated.

ECM contains many structural proteins:

Proteins provide structure and allow cell adhesion 1. Collagen fibers (90% of bone matrix protein content) 2. Elastin 3. Fibronectin

Proteoglycan versus Glycoprotein

Proteoglycan >95% carbohydrate Linked with protein via O-linked -Gal-Gal-Xyl-Ser (Exception keratan sulfate) Usually carbohydrate is unbranched Usually a specific repeating dimer of carbohydrates Glycoprotein Usually not more than 70% carbohydrate and often quite lower Linked with protein via O-linked -Man-Ser or N-linked through Asn Often branched carbohydrates Usually more variety in carbohydrates but often rich in mannose

ECM contains many proteoglycans:

Proteoglycans are highly charged protein/saccharide polymers (>95% carbohydrates) which prevent compression of ECM 1. Hyaluronic acid 2. Chondroitin sulfate 3. Dermatan sulfate 4. Heparan sulfate 5. Keratan sulfate

WHere does construction and degradation of proteoglycans occur?

Proteoglycans are primarily constructed in the Golgi apparatus The protein core is delivered from the ER and the carbohydrate is synthesized in the Golgi apparatus Separate enzymes are involved in each individual step Degradation of the proteoglycans occurs in the lysosomes Degradation occurs in a sequential manner with specific enzymes Disruption of a single enzyme in the sequence will result in accumulation of semidigested proteoglycans in the lysosomes

The large negative charge on proteoglycans leads to..

This large negative charge leads to binding of numerous counterions (cations) to neutralize the charge The high charge density leads to the influx of water into the ECM This results in swelling and stiffness of the ECM due to balancing of osmotic forces with binding interactions with proteoglycans and collagen

What are glycosaminoglycans (GAG)?

The carbohydrate portion of proteoglycans GAGs may contain up to 100 sugars per chain with specific repeating sequences composed of a uronic acid and an amino sugar These carbohydrates are polyanionic due to uronic acid and high level of sulfation on the amino sugars

You can think of the ECM as reinforced concrete support for the tissues

The collagen fibers act as rebar providing strength and support The proteoglycans act as an amorphous cement to tie everything together and provide rigidity

Proteoglycans are long chain carbohydrates. How do they provide the stiffness, rigidity and incompressibility to the ECM?

The large negative charge (which also has repulsive forces to each other) brings an influx of positive ions (cations) and water follows them into the ECM resulting in swelling and stiffness.

What is the main function of basement membrane?

The main function of basement membrane is to prevent migration of certain cell types between different tissue types

Functions of Proteoglycans

Their main role is to provide structural support to tissues, especially cartilage and connective tissue They are commonly responsible for preventing compression stress in tissues due to their large negative charges

What is the most common fibrillar collagen type found in the body?

Type 1 Collagen fibrils can be formed from a mixture of collagen types. Example: dermal collagen fibrils contain both Type I and Type III collagens

Which type of collagen makes up the basement membrane?

Type IV collagen is the primary structural component of basement membranes and forms a net-like structure

Why does scurvy affect so many tissues? For the tissues listed in the notes (slide 20), how would vitamin C deficiency cause the symptoms described?

Vitamin C is a cofactor (for all collagen synthesis) for the hydrolase enzymes which produce hydroxyproline. Vitamin C deficiency will lead to defective collagen synthesis. It is characterized by subcutaneous hemorrhages (strength lost in BVs), muscle weakness (muscle cells pulling against each other, if collagen is absent then muscles will pull themselves apart), soft swollen and bleeding gums (BVs there so mechanism similar to subcutaneous hemorrhages; collagen also holds teeth in and if damage collagen teeth will fall out), osteoporosis, poor wound healing and anemia.

What is Scurvy?

Vitamin C is a cofactor for the hydrolase enzymes which produce hydroxyproline Vitamin C deficiency will lead to defective collagen synthesis It is characterized by subcutaneous hemorrhages, muscle weakness, soft swollen and bleeding gums, osteoporosis, poor wound healing and anemia

Describe the function of glycine, proline and hydroxyproline to collagen structure. Why is osteogenesis imperfecta caused by a change in glycine position?

a. glycine - small (H-sidechain), which point inward into helix. allows for tight helical form. (tight packing of the helices) b. proline and hydroxyproline - are bent and rigid, allows for strength and bulk of structure. --> strength and rigidity are main functions. c. Osteogenesis Imperfecta - point mutation leads to a bulky replacement for gly, which disables the formation of helix. Collagen is no longer structurally sound.

Define Proteoglycan

are gel-forming portion of the ECM (sometimes called "ground substance" of the body, broader term than proteoglycan and includes other components). Contain protein chains with a large amount of complex carbohydrates attached (>95%).

What are Proteoglycans?

are the gel-forming portion of the ECM Sometimes called the "ground substance" of the body. Contain protein chains with a large amount of complex carbohydrates attached (Most are about 95% carbohydrate)

What are Basement membranes?

are thin layers of ECM that surround organs and generally provide a barrier between one tissue type and another

Extracellular Matrix (ECM) contains...

contains many fibrous proteins and proteoglycans

Interstitial fluid

is a complex aqueous soup of ions, proteins, proteoglycans and signaling molecules which surrounds all normal cells

What is Elastin?

is a protein which forms elastic fibers in blood vessels, lungs, ligaments and skin Primarily responsible for the elastic (stretchy) nature of the above tissues Elastin is formed as a single protein and has very little post-translational modifications

KS-I

is found primarily in the cornea and plays a role in cornea hydration Linked to asparagine via N-acetylglucosamine Disruption of keratan sulfate in the cornea is linked to macular corneal dystrophy

What is the main function of fibrillar collagens?

is to provide tensile strength to skin, tendons and ligaments

What are Aggrecans?

large macromolecular bottlebrush-like structures produced by the interaction of many different proteoglycans Collagen and other ECM proteins are embedded in this matrix

What are Fibrils are composed of?

many regularly overlapping triple helices of ~1000 amino acids in length The associate using both covalent (crosslinking) and non-covalent interactions

Nonfibrillar collagens interact with fibrillar collagens to produce what?

network or mesh-like structures