Biochemistry of Musculoskeletal system

PART-I Collagen Biosynthesis

Collagen
Collagen Biosynthesis

• Introduction

• Collagen and its types

Objective
• Collagen synthesis and steps

• Collagen synthesis disorders

What is collagen
Collagen is a fibrous proteins of ECM principally produced by fibroblasts cell

It is the main component of connective tissue & the most abundant protein in
mammals.

Collagen represent about 25% to 35% of the whole-body protein content.

 It is mostly found in connective tissue such cartilage, bones, tendons, ligaments,

and skin.

Collagen is also abundant in corneas, blood vessels, the gut, intervertebral discs,
and the dentin in teeth.
Major function of collagen

Collagen fibers support body tissues and collagen is a major component of the
extracellular matrix that supports cells.

Collagen and keratin give the skin its strength, waterproofing, and elasticity.

Loss of collagen is a cause of wrinkles in aged individuals.

Collagen production declines with age,

protein can be damaged by smoking, sunlight, and other forms of oxidative stress
Types of collagen
• The collagens can be organized into three groups, based on their structure and functions

1. Fibril-forming collagens:

Structure: Fibril-forming collagens assemble into long, rope-like structures called fibrils

• They composed of individual collagen molecules arranged in a staggered pattern.

Function: provide tensile strength and structural integrity to tissues.

They are abundant in tendons, ligaments, skin, and bones

Type I is the most abundant form & seen in connective tissues in almost all regions of the
body.
Types of collagen….
 Type I collagen fibers are found in high tensile strength like bone, tendon and cornea

 Type II is mainly seen in cartilage and vitreous humor,

 Type III found in lung and vascular tissues

2. Network-forming collagens:

Structure: create a mesh-like network and associate end-on to create sheet-like networks

Function: they play a crucial role in maintaining tissue architecture, cell adhesion, and
filtration. They are found in basement membranes
 Types IV and VII form a three-dimensional mesh, rather than distinct fibrils
 Type IV is seen in the basement membranes
Fibril forming
• These α chains are wound around one another in a rope-like triple helix
Network forming collagen
Collagen
3. Fibril-associated collagens:
Function: serve as connectors, bridging and attaching the collagen fibrils to each
other and to other ECM proteins.

Types IX and XII bind to the surface of collagen fibrils,

And linking these fibrils to one another and to other components in the
extracellular matrix
Types of collagen
Collagen
Collagen and its amino acids
• 33% of amino acid in collage is glycine, that is, every third residue is glycine.

• The repetitive amino acid sequence may be represented as Gly-X-Y-Gly -X–Y

• Every third amino acid is in close contact with the other two strands in the centre of the
structure

• Only glycine, which lacks a side chain, can fit in this position, and indeed, every third
amino acid residue of collagen is glycine

• Thus, collagen is a polymer of (Gly-X-Y) repeats, where Y is frequently proline or

hydroxyproline and X is any other amino acid found in collagen
Amino acids in collagens
• Proline facilitates the formation of the helical conformation of each α chain
because its ring structure causes “kinks” in the peptide chain

• Collagen contains hydroxyproline (hyp) and hydroxylysine (hyl), which are not
present in most other proteins
Hydroxylated amino acids in collagen

• Hydroxyproline residues are involved in hydrogen bond formation that helps to stabilize the triple
helix,

• whereas hydroxylysine residues are the sites of attachment of disaccharide moieties (galactose–
glucose)

• Hydroxylation require vitamin C (ascorbic acid) as a cofactor of the enzymes prolyl hydroxylase
and lysyl hydroxylase.

• In the absence of vitamin C (scurvy), the melting temperature of collagen drops from 42°C to
24°C

• This is because of the loss of interstrand hydrogen bond formation, which is in turn caused by the
lack of hydroxyproline residues.
Collagen synthesis
The collagen is synthesized by special cell called fibroblasts

The synthesis is take place both in intracellular and extracellular compartment

Collagen synthesis begin with activation of gene in the nucleus (COLA1-gene)

Each alpha chain poly peptide formed as the result of translation

The hydroxylation of proline, lysine residues and glycosylation of collagen is a

post-translational modification taking place intracellularly.
Synthesis cont.….

Prolyl hydroxylase and lysyl hydroxylase are used for hydroxylation of

polypeptide

These enzyme also contains ferrous iron at its active site and requires a reducing
agent

 Ascorbic acid used to preserve the iron in the reduced ferrous state

Lysyl oxidase and prolyl oxidase are cupper dependent enzyme used for cross-link
formation.
Intracellular synthesis of collagen
1. Formation of pre-pro-α chains:
 Collagen is one of many proteins that normally function outside of cells and produced for export.

 The newly synthesized polypeptide is called pre-pro-α chains

 Each per-pro polypeptides contain a special amino acid sequence at their N-terminal ends.

 This sequence is called signaling sequence.

2. Formation of pro-α chains

 This sequence acts as a signal to reach RER that the polypeptide being synthesized is destined to leave the
cell.

 The signal sequence is rapidly cleaved in the endoplasmic reticulum to yield a precursor of collagen called
a pro-α chain
Intracellular collagen synthesis

3.Hydroxylation
• The pro-α chains are processed by a number of enzymic steps within the lumen of the RER while
the polypeptides are still being synthesized.

• Proline and lysine residues found in the Y-position of the -Gly–X–Y– sequence can be
hydroxylated to form their hydroxy form
Intracellular collagen synthesis

4. Glycosylation: The hydroxyl group of the hydroxylysine residues of collagen may be

enzymatically glycosylated.

• Most commonly, glucose and galactose are sequentially attached to the polypeptide chain
prior to triple-helix formation
5. Assembly
• After hydroxylation and glycosylation, pro-α chains form procollagen

• The formation of procollagen begins with formation of interchain disulfide bonds between
the C-terminal extensions of the pro-α chains.

• This brings the three α chains into an alignment favorable for helix formation.
Intracellular collagen synthesis

6. Secretion
• The procollagen molecules are translocated to the Golgi apparatus, where they are
packaged in secretory vesicles.

• The vesicles fuse with the cell membrane, causing the release of procollagen
molecules into the extracellular space.
Extracellular process of collagen synthesis
7. Extracellular cleavage of procollagen molecules:
• After their release, the procollagen molecules are cleaved by N- and C-procollagen peptidases

• This remove the terminal propeptides, releasing triple-helical tropocollagen molecules.

8. Formation of collagen fibrils:
Individual tropocollagen molecules spontaneously associate to form collagen fibrils.
They form an ordered, overlapping, parallel array, with adjacent collagen molecules arranged
in a staggered pattern
Extracellular process of collagen synthesis
9. Cross-Links in Collagen Fibers
• The collagen fibers are strengthened by covalent cross-links between lysine and hydroxy lysine
residues.

• The cross-links are formed by lysyl oxidase which converts these amino acids into aldehydes.

• Lysyl oxidase is a copper containing enzyme, the copper ion being located at its active site.

• The reactive aldehydes that result (allysine and hydroxyallysine) can condense with lysyl or
hydroxylysyl residues in neighboring collagen molecules to form covalent cross-links and, thus,
mature collagen fibers

• In copper deficiency, reduced cross-linking of collagen results

Cross-Links in Collagen Fibers
Summary of collagen synthesis

1. Collagen synthesis start in the nucleus of fibroblast by protein translations

• The end product of this translated protein is called pre-pro-a-chain

• The pe-pro alpha chain has signaling peptide on the N-terminal that can be
recognized by rough ER.

2. At RER, the signaling peptide cleaved and form pro-alpha chain and secreted

3. The pro-alpha chains hydroxylated by lysyl and prolyl hydroxylase using O2 and
ascorbic acid as coenzyme
Summary of collagen synthesis
4. The hydroxylated three pro-alpha chains form disulfide linkage by their pro-
peptide at each end and form procollagen
5. Glycosylation
6. Modification
• The procollagen further modified in golgi apparatus to form vesicle and secreted
to ECM
7.In ECM the pro-peptides cleaved by peptidase enzyme and form fibril called
tropocollagen
8. Finally fibril cross-link formed by lysyl oxidase using CU2+ to form fibril
crosslink
 n c y
e fi ci e
e s i sd
s y nt h
l a g en
Co l
Scurvy
• In ascorbic acid deficiency affect the activity of hydroxylation enzymes, prolyl
hydroxylase & lysyl hydroxylase

• Inter chain H-bond formation is impaired formation of unstable triple helix

• Collagen fibrils cannot be cross-linked, greatly decreasing tensile strength of

assembled fiber

• Disease characterized by sore & spongy gums, loose teeth, fragile blood vessels,
swollen joints, & anemia
Scurvy
Osteogenesis Imperfecta

• Glycine founds in every third position of α- chain polypeptide

• R-group of glycine facilitates proper twisting of three α-chains during triple helix
formation

• Mutations in gene for Gly residues in pro-α1 or pro- α2 chains collagen cause
replacement of Gly residues (–Gly–X–Y–) by amino acids with bulky side chains

• Resultant structurally abnormal pro-α chains prevent formation of required triple-

helical conformation.
Ehlers-Danlos syndrome (EDS)

• Inherited deficiencies of extracellular collagen-processing enzymes

• Mutations in genes for amino acid sequences of collagen types I, III, or V

• Most clinically important mutations are found in gene for type III collagen is
potentially lethal vascular problems

• Collagen containing mutant chains is not secreted, & is either degraded or

accumulated to high levels in intracellular compartments

• Defects in collagen type I fibrils processing Fragile, stretchy skin & loose joints
Ehlers-Danlos syndrome (EDS)
Galatoomaa!
Quiz (10%)

1. What is the name of the cell that synthesizes collagen ?_____(1%)

2. Which type of collagen can be mostly affected in smoker individual?____(1%)
3. Which type of collagen found in the basement membrane of glomeruli of kidney
contain?___(1%)
4. The most dominant amino acid in collagen synthesis is called____(1%)
5. What are the two enzyme important for procollagen hydroxylation__(1%)
6. What is the cofactor/metal ion used for lysyl oxidase enzyme___(1%)
7. The type of collagen highly found in bone is called_____(1%)
8. Collagen disorder caused due to glycine gene mutation is______(1%)
9. Write the two posttranslational modification of collagen synthesis__(2%)
Biochemistry of skeletal system

PART-II Muscle energy metabolism

Biochemistry of skeletal muscle
Introduction
• Skeletal muscle, a remarkable tissue in our bodies, plays a crucial role in
movement, stability, and metabolism

• Human skeletal muscle consists of cells known as myocytes, also referred to as

muscle fibers

• Skeletal Muscle comprising about 40% of the average human body mass

• skeletal muscle consists of long, multinucleated fibers called muscle fibers.

• These fibers are grossly divided into two types

Types of muscle fibers
• Depending on their contraction speed and metabolic profile

Slow Twitch (Type I):

• These fibers primarily utilize fatty acid oxidation and contain abundant mitochondria.

• They store oxygen as oxymyoglobin, which gives them their reddish color.

• Contract slowely for long time and are energy efficient

Fast Twitch (Type II):

• Type IIa (intermediate fast twitch) and Type IIb (classic fast twitch) fibers:

• Type IIa: These fibers can use both aerobic and anaerobic metabolism for ATP production.

• Type IIb: These are the classic fast twitch fibers. Mainly use anaerobic metabolism

• Contract quickly and less energy efficient

Types of muscle fibers
How glucose inters into the cell

• Since glucose is polar and hydrophilic molecule it cannot diffuse directly to the cell
• There are a transport mechanism through which glucose get into the cells.
Transport mechanism

Na-independent facilitated diffusion Na-dependent glucose transport

They are designated GLUT-1 to GLUT-14 SGLT-1
No energy requiring ,down conc. Gradient Energy is required, is against conc.gradient
Glucose transportation to the cell

Facilitated diffusion of glucose via plasma membrane

Muscle cell glucose uptake
Fates of glucose in skeletal muscle
Glycogen Metabolism
Glycogen metabolism
• The constant source of glucose supply is absolutely required for cells.
• Glucose is the preferred energy source for brain and absolute source for mature
RBC.
• The three source of blood glucose are dietary intake of glucose, glycogen
degradation and gluconeogenesis
• Dietary intake of glucose in the form of complex CHO is sporadic and not reliable
Source of glucose.
• Gluconeogenesis have sustainability but slowly response to low glucose.
• Due to this our body has developed mechanisms for storing a supply of glucose in
a rapidly mobilizable form called glycogen
Glycogen metabolism cont.…

• The major store of glycogen in the body is skeletal muscle and liver.

• Muscle glycogen used to provide energy to muscle during exercise, not use for
other cells.

• Liver glycogen is to maintain blood glucose particularly during early fasting.

• 400g of glycogen stored in muscle and account 2% of muscle fresh weight

whereas 100g of glycogen store in liver and account 10% of liver weight.

• In glycogen storage disease the amount of store is high in both muscle and liver.
Glycogen structure

Picture taken from Lippincott illustrated 4th edition. Branched structure of glycogen, showing
α(1→4) and (1→6) linkages
Where is exact glycogen storage in cell

• Glycogen molecules exist in cytoplasmic granules associated with its degradatory

and synthetic enzymes.

• During early fasting liver glycogen depleted muscle glycogen depleted after
prolonged fasting.
Glycogen synthesis

• The process of glycogen synthesis called is also called glycogenesis

• Glycogen synthesis require primer for starting synthesis

• Primer is usually glycogen fragment or protein called glycogenin.

• Glycogenin is adding a few UDP-glucose until glycogen synthase enzyme

become active

• The process of adding UDP-Glucose without enzyme is called autoglucosylation.

Primer
Steps in glycogenesis

Step-1 Glucose phosphorylation and isomerization

G-6-P is converted to G-1-P by phosphoglucomutase enzyme .

Step-2 glucose activation. G-1-P + UTP------UDP-Glucose + PPi by UDP-glucose pyro phosphorylase

.
Step in glycogenesis cont.…

Step-3 Primer synthesis.

• The glycogen primer is formed by autoglucosylation of glycogenin.

• Glycogenin is a dimeric protein, the monomers glycosylating each other using

UDP-glucose till seven glucose units are added.

Step-4 Elongation of glycogen by glycogen synthase.
Glycogenesis

Step-5 formation of branch

The amylo-α(1→4) → α(1→6)-trans glucosidase enzyme first cut 8 glucosyl
residues from the long glycogen.

 Then it form branch with new nonreducing end.

Then after, elongation of branch by glycogen synthase

Glycogenesis

The enzyme amylo- 1, 4→1, 6-transglucosidase

(branching enzyme) forms the alpha-1, 6 linkage

Adopted from Textbook of biochemistry for medical student

Glycogen degradation (glycogenolysis)

• Glycogen degradation is the mobilization of stored glucose in liver and muscle.

• It is not the reverse of glycogen synthesis as it involve different enzyme.

Step-1 shortening

• The long glucose shortened by glycogen phosphorylase

• This enzyme use pyrophosphate as coenzyme and cleave 1-4 bond until 4 glucosyl
residues remain before branch of 1-6.

• This short structure called limit dextrin.

Cont..

• Step-2 branch removing.

• The three glucose before branch is removed out and attached to another
nonreducing end

• The enzyme called amylo-α(1→4) → α(1→4) glucan transferase(4:4 transferase).

• The remaining branching glucosyl residue removed by amylo-α(1→6) glucosidase

to free glucose.

• These two enzyme called debranching

Glycogenolysis summary
Summary of Glycogenesis vs Glycogenolyis
Glycogenolysis of liver cell

• In the liver, glucose 6-phosphate is translocated into the endoplasmic reticulum (ER) by glucose 6-
phosphate translocase.

• There it is converted to glucose by glucose 6-phosphatase—the same enzyme used in the last step
of gluconeogenesis.

• The resulting glucose is then transported out of the ER to the cytosol by GLUT-7.

• Hepatocytes release glycogen-derived glucose into the blood to help maintain blood glucose levels
until the gluconeogenic pathway is actively producing glucose.
Difference of glycogenolysis in liver and
muscle
Glycogenolysis
Glycogen metabolism regulation
• In order to maintain blood glucose, glycogen synthesis and degradation should be tightly regulated

• Glycogen synthesis in liver accelerated during well fed and degradation accelerated during fasting.

• In muscle degradation accelerated during exercise and synthesis after exercise/at rest.

1.Allosteric regulation
• Both glycogen synthase and glycogen phosphorylase regulated by the metabolites and energy
level of the cell

• Well fed state/The availability of substate like G-6-P and high energy(ATP) allosterically
activate glycogen synthase enzyme.
Glycogen metabolism regulation cont.…

Taken from Lippincott illustrated 4th ed. Allosteric regulation of glycogen synthesis and
degradation.
Glycogen metabolism regulation

• The presence of G-6-P and high ATP inhibit glycogen phosphorylase

2. Glycogen degradation activation in muscle by Ca++
 During muscle contraction energy is needed as ATP for the muscle cells

 This energy is supplied by degradation of stored glycogen by muscle. How?

 Nerve impulse cause muscle membrane depolarization that cause the release of Ca ++ from muscle
sarcoplasm reticulum to cytoplasm of muscle.

 The released muscle bind to calmodulin(4Ca++-calmodulin complex) formed.

 This complex bind to protein kinase in cytoplasm w/c is Camp-independent kinase.

Muscle glycogen phosphorylase regulation by Ca++

• Activated protein kinase phosphorylate glycogen phosphorylase

• Then, muscle glycogen breakdown enhanced and G-6-Pfor ATP released

• When muscle relax released Ca++ returned to sarcoplasmic reticulum and kinase become inactive

.
Glycogen metabolism regulation cont..

2. cAMP-dependent glycogenolysis regulation

 Hormone like glucagon and catecholamine release during fasting bind to their specific membrane
receptor.

 When they bind they activate receptor and then G-couple receptor finally cAMP increase

 cAMP activate cAMP-dependent protein kinase through binding to its R-subunit

 Activated cAMP-dependent protein kinase phosphorylate glycogen phosphorylase(inactive-b-form)

to active-a form.
Stimulation and inhibition of glycogen degradation
Hormonal regulation of Glycogen synthase
Glycogen storage diseases
Testing understand

• The energy yield from one glucose residue derived from glycogen is 3 ATP
molecules, why?
Cori cycle
Cori cycle

• Cori cycle is also known as lactic acid cycle, discovered by Carl Cori and Gerty Cori.

• Lactate released from cells undergoing anaerobic glycolysis is taken up by other tissues (primarily
the liver, heart, and skeletal muscle) and oxidized back to pyruvate.

• In the liver, the pyruvate is used to synthesize glucose (gluconeogenesis), which is returned to the
blood

• The cycling of lactate and glucose between peripheral tissues and liver is called the Cori cycle
Cori cycle cont.…

• Muscular activity requires ATP, which is provided by the breakdown of glycogen in the skeletal
muscles

• The breakdown of glycogen, releases glucose in the form of glucose 1-phosphate (G-1-P).

• The G1P is converted to G-6-P by phosphoglucomutase.

• G-6-P is readily fed into glycolysis to provide muscle immediate energy in activity.

• During muscular activity, the store of ATP needs to be constantly replenished.

• However, O2 supply reduced during intensive muscular activity(hypoxia)

Cori cycle cont.…

• In such condition, lactic acid fermentation converts pyruvate to lactate by lactate dehydrogenase.

• Most importantly, fermentation regenerates NAD+, maintaining its concentration so additional

glycolysis reactions can occur.

• The lactate formed in this reaction released to circulation and taken by liver.

• Liver convert lactate to free glucose via gluconeogenesis and release back to muscle through
circulation.

• If muscle activity has stopped, the glucose is used to replenish the supplies of glycogen through
glycogenesis
Cori cycle cont.…
Significancy of Cori cycle

• Prevent lactic acidosis in skeletal muscle

• Is source of energy during exercise

• Cori cycle is important source of substrate for gluconeogenesis.

Is Cori cycle sustainable work?

 Overall, the glycolysis steps of the cycle produce 2 ATP molecules at a cost of 6

ATP molecules consumed in the gluconeogenesis steps. Due to its energy cost Cori cycle is not sustainable
indefinitely.