Contents 1 Osteogenesis Imperfecta 2 Learning Objectives 3 Collagen 3.1 Structure and Function 3.2 How is it synthesized? 3.2.1 Signaling Pathway 3.3 Different types? Where are they found? What are its functions? 3.4 How is collagen important in bone integrity and health? 4 Amino Acids 4.1 Structures, especially glycine and valine 5 Pathophysiology of Osteogenesis Imperfecta 5.1 Why is her sclera blue-grey? 5.1.1 Differential Dxs 5.2 Most common mutations 5.3 Col1A1 gene 5.4 Why L1 fracture specifically? Random? 5.5 Treatment options 6 Other diseases resulting from collagen deficiency 7 Miscellaneous 7.1 Protein Folding 7.2 De novo mutations 8 Useful Resources

[edit] Osteogenesis Imperfecta

Goal: The goal of this case is to understand the structure of collagen and how this protein's functions are determined by its structure.

[edit] Learning Objectives

1. List the 4 primary types of collagen and describe their location and function. Type Chain composition Representative Tissues I a1(I)2 a2(I) Skin, bone, tendon, BVs, cornea II a1(II)3 Cartilage, intervertebral disk, vitreous body III (reticular fibers) a1(III)3 BVs, fetal skin, basal lamina IV a1(IV)2 a2(IV) BM

2. Describe the unique characteristics of the collagen family of proteins (amino acid sequence, secondary structure, quaternary structure).

• 1*: Gly-X-Y where X = Pro, Y = hydroxyPro or hydroxyLys
• 2*: a-helix
  • Hydroxylation of Pro, Lys = posttranslational mod.
  • HydroxyLys may be glycosylated w/ Glu or Gal prior to triple helix formation
• 4*:
  • Triple helix (3 a-chains wrapped around each other); held together by H-bonds b/t chains
    • Flanked by non-helical NH2- & COOH-terminal extensions, which participate in interchain disulfide bonds with NH2- & COOH-terminal extensions of other a-chains

OUTSIDE OF CELL

• • Spontaneous fibril formation
    • Ordered, overlapping, parallel array w/ adj. collagen molcs arranged in staggered pattern

3. Explain how special structural features of proline and glycine are required for their roles in the collagen triple helix.

• Glycine is the smallest aa, so it can fit into tight spaces.
• Proline’s side chain is bonded to both the nitrogen & Ca. This ring structure makes it more conformationally restricted, putting kinks in collagen structure.

4. Draw a diagram listing the steps in the assembly of collagen from the DNA to the finished protein secreted from the cell. 5. List the role and function of post-translational modifications and crosslinks (inter- and intrachain) in collagen.

• Post-translational mod
  • Hydroxylation of Pro, Lys → stable helical configuration: form interstrand H-bonds, proper cross-link form’n, proper secretion from fibroblast
  • Glycosylation of hydroxyLys (w/ Glu or Gal) prior to triple helix formation
• Cross-links → necessary for tensile strength → proper f’ning
  • b/t lysyl & hydroxylysyl res of adj. collagen molcs

6. Describe the role of vitamins in collagen formation.

• Vitamin C reduces the ferric ion of the inactivated enzyme → now active; Vit C = antioxidant
• Collagen synthesized in absence of Vit C is less stable: hydroxyPro stabilizes collagen triple helix by forming interstrand H-bonds

7. List other examples of human genetic diseases that are caused by mutations in collagen (Marfan's Syndrome and Ehlers-Danlos Syndrome).

• Ehlers-Danlos syndrome = causes progressive deterioration of collagens, w/ diff. EDS types affecting diff. sites in body i.e. joints, heart valves, organ walls, arterial walls
• Osteogenesis imperfecta
• Marfan syndrome

• 1. List the 4 primary types of collagen and describe their location and function.
• 2. Describe the unique characteristics of the collagen family of proteins (amino acid sequence, secondary structure, quaternary structure).
• 3. Explain how special structural features of proline and glycine are required for their roles in the collagen triple helix.
• 4. Draw a diagram listing the steps in the assembly of collagen from the DNA to the finished protein secreted from the cell.
• 5. List the role and function of post-translational modifications and crosslinks (inter- and intrachain) in collagen.
• 6. Describe the role of vitamins in collagen formation.
• 7. List other examples of human genetic diseases that are caused by mutations in collagen (Marfan's Syndrome and Ehlers-Danlos Syndrome).

[edit] Collagen

[edit] Structure and Function

Collagen is rich in proline and glycine. Proline causes "kinks" in the peptide chain, which faciliate the formation of the helical confirmation of the alpha chain. Glycine is found as every 3rd residue of the chain in a repeating sequence: -Gly-X-Y-, where X is usually proline and Y is usually hydroxyproline or hydroxylysine. The hydroxylation of proline and lysine is an example of posttranslational modification. Hydroxyproline is important in stabilizing the helical structure of collagen because it maximizes interchain hydrogen bond formation. Interchain disulfide bonds help initiate helix formation. Hydroxylysine may be glycosylated. Bands: light staining where no gaps, dark staining with gaps

[edit] How is it synthesized?

1) Cytosol

Collagen alpha-chain synthesis starts in the cytosol, where the amino terminal signal sequence binds signal recognition particles. Hydroxylation of proline and lysine residues occurs cotranslationally. Proline hydroxylation stabilizes collagen, and lysine hydroxylation provides sites for interchain cross-linking and for glycosylation.

Triple helix assembly occurs after the polypeptide chains have been completed. Carboxy-terminal globular proprotein domains fold and disulfide bonds are formed between them. This initiates winding of the triple helix from the carboxyl- toward the amino-termini.

2) Golgi

The completed triple helix, with globular proprotein domains at each end, moves to the Golgi apparatus where oligosaccharides are processed and matured.

3) Released from cell

The completed procollagen is then released from the cell via secretory vesicles.

4) Collagen Maturation

Conversion of procollagen to collagen occurs extracellulary. The amino- and carboxyl-terminal propeptides are cleaved by separate proteases. The triple helices assemble into fibril by end to end and side to side aggregation, and the collagen is stabilized by cross-linking. Lysyl oxidase converts some lysine of hydroxylysine to the reactive aldehydes causing the residues to condense with each other or with adjacent chains to form Schiff’s base and aldol cross-links.

Source: DEVIN Biochemistry

NJK

Additional details regarding synthesis: The propeptides that are cleaved to convert procollagen to collagen have two functions: one is to guide the intracellular formation of the triple-stranded collagen molecules, the other is to prevent the intracellular formation of collagen fibrils. Also, individual collagen molecules interact spontaneously to form fibrils in an ordered, overlapping, parallel array. the adjacent molecules are staggered, overlapping one another by about three-quarters of a molecule. Fibroblasts “tug” the collagen fibrils into regular positions. Half life: several months, but in response to injury or growth, will be shorter

Source: Alberts, The Cell; Lippincott's Biochem Review

ASR

I like pictures.

The Cell

JT

[edit] Signaling Pathway

Fig. 4. Diagram of the signaling pathway involving LRP5. (A): Wnt binds to its Frizzled receptor and LRP5 co-receptor, activating the canonical signaling pathway and thereby increasing bone formation. (B): Dkk (Dickkopf-1) binds to its Kremen receptor and LRP5 co-receptor, preventing Wnt from binding to LRP5 and thereby blocking the canonical pathway and inhibiting bone formation. (C): The G171V LRP5 mutation prevents Dkk from binding to LRP5, thus facilitating the binding of Wnt to LRP5 and the activation of the canonical pathway, the result being increased bone formation.

Remember this?

LRP5 is located on osteoblast membranes. Activation of this pathway leads to an increase in bone formation.

http://journals.ohiolink.edu/cgi-bin/sciserv.pl?collection=journals&journal=1297319x&issue=v72i0003&article=207_lmiosahd&form=fulltext

This pathway is not disrupted in osteogenesis imperfecta.

JT

[edit] Different types? Where are they found? What are its functions?

There are more than 20 collagen types. Some more common types and their locations:

Fibril-forming collagens: Types I, II, and III have rope-like structures and characteristic banding patterns reflecting the regular staggered packing of the individual collagen molecules in the fibril.

Type I

• Skin
• bone
• tendon
• blood vessels
• cornea

Type II

• Cartilage
• Intervertebral disk
• Vitreous body

Type III (reticular fibers)

• Blood vessels
• Fetal skin

Network-forming collagens: Types IV and VII form a three-dimensional mesh as opposed to distinct fibrils.

Type IV

• Basement membrane

Type VII

• Beneath stratified squamous epithelia

Fibril-associated collagens: Types IX and XII link collagen fibrils by binding to their surfaces in the extracellular matrix.

Type IX

• Cartilage

Type XII

• Tendon
• Ligaments

Collagen types can be divided into different classes:

• Fibrillar collagen: Characterized by long, stiff, triple helices cross-linked in a staggered array. Type I (skin), Type II (cartilage), Type 3 (arteries).
• Fibril-associated collagen: Characterized by fibrils linked to other components in the extracellular matrix. Type IX (cartilage), Type XII (ligaments).
• Network collagen: Characterized by assembly into sheet or meshwork. Type IV (basement membrane), Type VII (found beneath squamous epithelium).

Source: Lippincott's Biochemistry

MDM

[edit] How is collagen important in bone integrity and health?

• Half life: in bone, could be up to 10 years
• Collagen and bone protein are needed to make bone strong
• Hydroxyapatite is secreted into the collagen fibers by osteoids

[edit] Amino Acids

[edit] Structures, especially glycine and valine

Critical amino acids are proline, lysine, and glycine.

Proline: Cyclic structure makes it a good choice to place at bends in protein structure. The thermodynamic forces that cause the formation of the helix structure in collagen are driven by the cyclic structure of proline.

Lysine: Hydroxyl lysine is modified to create interchain N-C covalent bonds

Glycine: Possesses the smallest side group of any amino acid which allows it to pack tightly into the protein structure. Allows for the close packing of the collagen alpha chains into the polypeptide structure of the collagen molecule.

Valine: no special significance per se, but the side chain is much larger than that found on glycine, thus preventing the close packing of the alpha chains into the polypeptide structure.

Source: Devlin biochem

NCB

The amino acid sequence differs for the different types of collagen, but a repeating Gly-X-Y sequence of about 1000 residues predominates. Every third residue is glycine, about one third of the X positions are occupied by proline, and a similar number of Y positions are 4-hydroxyproline, a posttranslationally modified form of proline.

Source: DEVIN Biochemistry NJK

[edit] Pathophysiology of Osteogenesis Imperfecta

[edit] Why is her sclera blue-grey?

The deficiency in collagen production makes her sclera more translucent which allows partial visualization of the underlying choroid.

Robbins

JT

Sclera

• White portion of eye.
• Composed of dense regular collagenous connective tissue that provides attachment for the extrinsic muscles of the eye.
• Normally appears white. Appears blue in OI because of collagen loss of the sclera, resulting in partial visualization of the deepest layer of the sclera. This enables light to reflect off of the melanin produced by melanocytes in the suprachoroid lamina. Melanin absorbs long wavelengths of light and scatters short wavelengths (Tyndall effect), which make the sclera appear bluish.
• This melanin effect is also seen in blue nevi on the skin. Image: http://www.dartmouth.edu/~thabif/weeklyclinic100101/22bluenevus.html

Sources: Gartner, Color Atlas of Histology; eMedicine "Blue Nevi"

MDM

[edit] Differential Dxs

Blue sclera may appear in a variety of conditions:

• Staphyloma: In eyes with exceptionally high intraocular pressure, the sclera may become thin and appear blue clinically. Image: http://www.yemenweb.com/algorafi/cornea1.jpg
• Congenital melanosis oculi: Heavily pigmented congenital nevus (benign mole) of the underlying uvea (pigmented layers of the eye that contain most intraocular blood vessels).
• Nevus of Ota: Melanosis oculi accompanied by pigmentation of the periocular skin. Image: http://images.google.com/images?hl=en&q=Nevus%20of%20Ota&ie=UTF-8&oe=UTF-8&sa=N&tab=wi
• Melanoma of the eye

Source: Robbins and Cotran

MDM

[edit] Most common mutations

Osteogenesis Imperfecta Type 1 (Osteogenesis imperfecta tarda)

• Frameshift mutations, insertion of premature stop codons result in decrease synthesis of the pro-alpha 1 chain, resulting in decreased collagen formation. Less severe than type 2.

Osteogenesis Imperfecta Type 2 (Osteogenesis imperfecta congenita)

• Missense mutations resulting in the substitution of glycine for another amino acid with a larger side chain. Prevents close packing of alpha chains into polypeptide structure of collagen (dominant negative effects).

In both type 1 and 2 mutations are ususally autosomal dominant.

Type 4 has some bone deformity and abnormal collagen formation (patient may have this) Type 1 has decreased collagen quantity but collagen normal Type 3 has extremely short stature

Mutations in the C terminus are more disruptive

Source: Robins Pathology, Thompson Genetics

NCB

[edit] Col1A1 gene

Located on chromosome 17q21.31-q22. Codes for alpha type 1 (I) chain.

[edit] Why L1 fracture specifically? Random?

http://www.spine-health.com/Topics/case/kyph/kyphoplasty01.html

The image is a compression fracture of the L1 vertebrae. As far as I can tell, the lumbar vertebrae bear more weight than any other vertebrae, and L1 is the least substantial of the five.

http://www.spineuniversity.com/images/DPUSA227_CFracture/images2/Image_08b_DPUSA227.gif

A compression fracture is an injury incurred by the spine when pressure causes a vertebrae to be crushed into a smaller volume than it should be, as shown above. Compression fractures occur most commonly where L1 and T12 meet.

http://www.dynomed.com/encyclopedia/encyclopedia/spine/Compression_Fracture.htm

JT

[edit] Treatment options

4 main goals of treatment:

• Reduce fracture rates
• Decrease long bone deformities in scoliosis
• Minimize chronic pain
• Maximize mobility and other functional capabilities

Treatment is mainly responsive, that is, orthopedic surgery as needed. Bisphosphonates may be used to inhibit bone resorption. These compounds have been used to treat a number of bone diseases, such as osteoporosis and Paget's. Limited success is evident for OI.

Lange Endocrinology

Bisphosphonates work by binding to the bone matrix. Osteoclasts that attempt to resorb the bone also phagocytose the bisphosphonate which kills them.

Wikipedia

JT

[edit] Other diseases resulting from collagen deficiency

Deficiency in type 2 collagen (located in cartilage and intervertebral disks):

Achondrogenesis

• A group of severe disorders characterized by a small body, short limbs, and other skeletal abnromalities.
• Infants are usually born prematurely, are stillborn, or die shortly after birth from respiratory failure.
• Incidence: 1 in 40,000 to 60,000 births

Source: NLM Genetics Home Reference, http://images.google.com/images?hl=en&q=achondrogenesis&btnG=Search+Images

Deficiency in type 9 collagen (stabilizes network of cartilage type 2 collagen fibers):

Multiple epiphyseal dysplasia

• Characterized by skeletal deformations resulting from impaired endochondral ossification and premature degenerative joint disease
• This condition has relatively mild signs and symptoms, including joint pain that usually begins in childhood; malformations of the hands, feet, and knees; and abnormal curvature of the spine (scoliosis).
• Incidence unknown. Some affected people may never be diagnosed because the features of the condition are often mild.

Source: NLM Genetics Home Reference, Ross Histology, Robbins and Cotran

Deficiency in type 10 collagen (contributes to bone mineralization, produced by chondrocytes in normal growth plate):

Schimid metaphyseal chondrodysplasia

• Characterized by short stature with abnormally short arms and legs (short-limbed dwarfism) and bowed legs.
• Other physical characteristics may include outward flaring of the bones of the lower rib cage and/or hip deformities in which the thigh bone is angled toward the center of the body, typically resulting in an waddling gait.
• Incidence: very rare

Source: WebMD.com, Ross Histology, Robbins and Cotran

Deficiency in multiple collagen genes:

Ehlers-Danlos Syndrome

• 6 variants have been recognized. Locus heterogeniety occurs.
• Characterized by hyperextensible skin, tendency to bleed, easy bruising, and hypermobile joints.
• Defects in Type 1 collagen fibrils result in stretchy skin and loose joints.
• Most clinically important mutations are in the gene for Type III collagen. Potentially lethal vascular problems occur.
• Incidence: 1 in 5000 to 250,000 (varies by EDS type)

Source: NIH Genetics Home Reference, Robbins and Cotran, Lippincott Biochemistry

MDM

Marfans: problem with fibrillin

[edit] Miscellaneous

[edit] Protein Folding

• Primary: the sequence of the amino acids (most simple)
• Secondary: refers to certain common repeating structures found in proteins. There are two types of secondary structures: alpha-helix and beta-pleated sheet. Mostly involves hydrogen bonding
• Tertiary: the full 3-dimensional folded structure of ONE polypeptide chain.
• Quaternary: the interaction and connections between multiple polypeptide chains (subunits)

[edit] De novo mutations

De novo mutations happen to one gamete as opposed to germline mutations where all gametes are affected. This one gamete mutation produces the founder of the disease.

[edit] Useful Resources