BEU11403-Biomaterials Chapter 2: Assembly of Biological Macromolecules

Chin Fhong, Soon (PhD, MIET, MIEEE-EMBS) Microelectronics and Nanotechnology-Shamsuddin Research Center

Lecture outline
Collagen Fibronectin Laminin Fibrinogen Glycoproteins

Basement membrane

Collagen
Major

insoluble fibrous protein of the extracellular matrix and connective tissue Most abundant protein in animals Made by fibroblasts and some epithelial cells tensile strength and elasticity
Tendons Cartilage Bone

half

total body proteins (by weight)

Collagen

Collagen Components
Triple helix of polypeptides Each polypeptide is a left-handed helix too Insoluble glycoprotein Protein

protein + carbohydrate

high glycine high proline hydroxylysine hydroxyproline (gly-X-Y)

Carbohydrate, glucose, galactose

Extracellular Matrix contains proteins collagen, elastin, fibronectin, and laminin

Collagen is strong, resisting tensile forces (in abundance in the Achilles heel)

Y is usually hydroxyproline

Collagen

Collagen Structure
Collagen Protein 3 polypeptide (a) chains, left hand helix, forms fibers many different (vertebrate) collagens by different combinations of -chains Type I, II, III

main fibers, flexible

bone, skin, tendons 90% of all collagen cartiladge

Type I

Type II

Type I, II, III cross striated

Collagen Fibers

Type IV fine unstriated

e.g. tendons - type I fibrils, have a 67-nm period striations and are oriented longitudinally (direction of the stress) showing overlapping packing of individual collagen molecules reticular fibres type III, support individual cells

Type V-XII

sheet-like supportive meshwork mature basal laminae tracks for embryonic migration barriers for cell migration
smaller diameter fibers than I-III no striations

Collagen Type Function

Collagen Type I - skin, tendon, vascular, ligature, organs, bone (main component of bone) Collagen Type II - cartilage (main component of cartilage) Collagen Type III - reticular fibers with type I. Collagen Type IV - forms bases of cell basement membrane Collagen Type V - hair and nail

PhD Research Results

Figure 3.12. Micrographs of immuno-staining against collagen type IV, laminin and fibronectin for HaCaT cells cultured on (a, d, g) negative controls,(b, e, f)controls and (c, f, i) liquid crystal substrates, respectively. (Scale bar: 20 m). (Enlarged exert, scale bar: 25 m).

Staining of Laminin
Negative control Control

Figure 3.13. Immunoperoxidase staining of laminin for HaCaT cells cultured on a negative control (left), control (middle) and liquid crystal substrate (right).

Endoplasmic Reticulum

Collagen Synthesis

Golgi Apparatus

mRNA attached to ER protein synthesized into ER lumen cotranslational and post-translational modifications 3 proto--chains form soluble procollagen moved to golgi apparatus packed into secretion vesicles fuse with membrane procollagen processed by enzymes outside cell assemble into collagen fibers collagen fibrils form lateral Interactions of triple helices

Outside Cell

Co and Posttranslational modification (PTM) is a step in protein biosynthesis. Proteins are created by ribosomes translating mRNA into polypeptide chains. These polypeptide chains undergo PTM, (such as folding, cutting and other processes), before becoming the mature protein product.

Collagen synthesis

Elastin
Elastic

fiber is composed of the protein fibrillin and elastin made of simple amino acids such as glycine, valine, alanine, and proline. Elastin lacks collagens repetitive sequeencing at the fibril stage. Fibres are only 5 to 7 mm in length. High hydrophobic and elastic.

Fibronectin

Fibronectin
Dimer

S-S

linkages rigid and flexible domains fibronectin fibrils have elastic properties and can stretch fibrils up to four-fold their relaxed length. fibrillogenesis - transformation from the compact (soluble) form to the extended fibrillar (insoluble) form of fibronectin, requires application of mechanical forces generated by cells.

MW 440 kDa nearly identical subunits composed of types I (F1), II (F2), and III (F3) fibronectin modules

connected at C-terminus

Two types of fibronectin are present in vertebrates:[ soluble plasma fibronectin (formerly called "coldinsoluble globulin", or CIg) is a major protein component of blood plasma (300 g/ml) and is produced in the liver by hepatocytes. insoluble cellular fibronectin is a major component of the extracellular matrix. It is secreted by various cells, primarily fibroblasts, as a soluble protein dimer and is then assembled into an insoluble matrix in a complex cell-mediated process.

Fibronectin is a high-molecular weight (~440kDa) glycoprotein of the extracellular matrix that binds to membrane-spanning receptor proteinscalled integrins. Similar to integrins, fibronectin binds extracellular matrix components such as collagen, fibrin, and heparan sulfateproteoglycans (e.g. syndecans). Fibronectin exists as a protein dimer, consisting of two nearly identical monomers linked by a pair of disulfide bonds.[1] The fibronectin protein is produced from a single gene, but alternative splicing of its premRNA leads to the creation of several isoforms.

cell

Binding Domains
arg-gly-asp-ser

binding segment RGDS

binds

domains

then mechanically couples to the actin cytoskeleton

integrin receptor in membrane bind

heparin sulphate collagen hyaluronic acid Gangliosides fibronectin

Fibronectin Function
soluble

insoluble

blood clotting process, link to fibrin

protein in blood plasma (200250 kDa monomer)

ECM fibronectin differs from plasma fibronectin by the presence of additional polypeptide segments and in altering morphology of transformed cells and hemagglutination.

protein in extracellular matrix (ECM)

Cell Adhesion Migration Pathways blocking fibronectin with antibody prevents neural crest migration extension of axons and dendrites branching

Fibronectin plays a major role in cell adhesion, growth, migration, and differentiation, and it is important for processes such as wound healingand embryonic development. Altered fibronectin expression, degradation, and organization has been associated with a number of pathologies, including cancer and fibrosis.

Laminin

Laminin Structure
cross-shaped

glycoprotein 3 polypeptides , 1, 2 carbohydrate (13% by weight) separate binding domains

collagen IV heparin heparin sulphate cell binding cell specific binding - liver, nerve cell surface receptor

Laminins are major proteins in the basal lamina (one of the layers of the basement membrane), a protein network foundation for most cells and organs. The laminins are an important and biologically active part of the basal lamina, influencing cell differentiation, migration, and adhesion, as well as phenotype and survival

Laminin Function
cell

adhesion migration pathways stimulates growth of axons development and regeneration differentiation basal laminae most abundant linking glycoprotein Integrin- Structure Integrin Function cell membrane receptor for ECM linkers binds RGDS. Integrins consists of 2 subunits alpha () and beta () transmembrane linked to cell cytoskeleton actin microfilaments via talin and vinculin focal contacts
For

Review see Integrin signaling revisited. Schwartz MA. Trends Cell Biol 2001 Dec;11(12):466-70

Actin filament
Cell

ECM

Actin filament -actinin

Capping protein

Vinculin paxillin Talin

Cytoplasm

Integrin receptors Bilayer lipid membrane

Laminin protein

The structures of actin cytoskeleton, focal adhesion complexes, integrin receptors, and adhesion proteins.

Fibrinogen

Fibrinogen
is a soluble, 340 kDa plasma glycoprotein, that is converted by thrombin into fibrin during blood clot formation. Fibrinogen is synthesized in the liver by the hepatocytes. The concentration of fibrinogen in the blood plasma is 200400 mg/dL.

Glyco proteins - a protein with a carbohydrate attached to it. More protein to carbohydrate. eg: enzymes, hormones, cell membrane components.

Glycoproteins

Proteo glycans - Disaccharide chains attached to a protein core. Rem - glycan refers to glucosamino glycans. More carbohydrates than proteins. (95% to 55%) eg: heparin, heparan, keratan, dermatan, chondroitin

Proteoglycans

Glycoproteins
Glycoproteins are proteins that contain oligosaccharide chains (glycans) covalently attached to polypeptide side-chains. The carbohydrate is attached to the protein in a cotranslational or posttranslational modification. This process is known as glycosylation. Secreted extracellular proteins are often glycosylated. In proteins that have segments extending extracellularly, the extracellular segments are also glycosylated. Glycoproteins are often important integral membrane proteins, where they play a role in cellcell interactions. Glycoproteins are also formed in the cytosol, but their functions and the pathways producing these modifications in this compartment are less well understood

Glycoaminoglycans
Glycosaminoglycans (GAGs) or mucopolysaccharides are long unbranched polysaccharides consisting of a repeating disaccharide unit. The repeating unit (except for keratan) consists of an amino sugar (N-acetylglucosamine or Nacetylgalactosamine) along with a uronic sugar(glucuronic acid or iduronic acid) or galactose.[3] Glycosaminoglycans are highly polar and attract water. They are therefore useful to the body as a lubricant or as a shock absorber.

Glycosylation
Glycosylation (see also chemical glycosylation) is the reaction in which a carbohydrate, i.e. a glycosyl donor, is attached to a hydroxyl or other functional group of another molecule (a glycosyl acceptor). In biology glycosylation mainly refers in particular to the enzymatic process that attaches glycans to proteins, lipids, or other organic molecules. This enzymatic process produces one of the fundamental biopolymers found in cells (along with DNA, RNA, and proteins). Glycosylation is a form of co-translational and post-translational modification. Glycans serve a variety of structural and functional roles in membrane and secreted proteins. The majority of proteins synthesized in the rough ER undergo glycosylation. It is an enzyme-directed sitespecific process, as opposed to the non-enzymatic chemical reaction of glycation

The end of chapter 2.