Fibronectin

1
Fibronectin
Hitaa Gandhi, BarShaina Lamar, Hardik Modi
2
Background

• Is involved in many cellular processes
• It exists in two (2) main forms
• Insoluble glycoprotein dimer
• Soluble disulphide linked dimer
• A high-molecular glycoprotein
• Serves as a general cell adhesion molecule.

3
Fetal Fibronectin

• Fetal Fibronectin (fFN) is a protein produced by
  fetal cells.
• Found at the interface of the chorion and the
  decidua.
• Is an excellent biological marker of premature
  delivery.

4
Structure Background

• Contains the Arg-Gly-Asp (RGD) the FN receptor
  (integrin) binding motif
• It is a ligand to at least 13 different
  integrins.
• It is composed of three different types of
  homologous, repeating modules
• Type I
• Type II
• Type III

5
Structure
III7
III7
III7
III7
III8
III8
III8
III8
III9
III9
III9
III9
III10
III10
III10
III10
http//bioscience.org/1997/v2/d/johanss1/htmls/3.h
tm
6
http//bioscience.org/1997/v2/d/johanss1/htmls/3.h
tm
The Mechanism

• RGD is located on the Module 10
• Integrin binds to the unknown site
• FN 1-5 binds to the unknown site.

7
Structure

• 1 Chain
• 368 amino acids
• 4,220 atoms

8
Hydrophobic Core
9
ChouFas
alpha helices
368 AA Beta Sheet alpha helices turns
10
Gor4
PLSPPTNLHLEANPDTGVLTVSWERSTTPDITGYRITTTPTNGQQGNSLE
EVVHADQSSCTFDNLSPGLEYNVSVYTVKDDKESVPISDTIIPAVPPPT
D LRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNEEDVAELSISPSD
NA VVLTNLLPGTEYVVSVSSVYEQHESTPLRGRQKTGLDSPTGIDFSDI
TAN SFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNL
TPGT EYVVSIVALNGREESPLLIGQQSTVSDVPRDLEVVAATPTSLLIS
WDAPA VTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTI
TVYAVT GRGDSPASSKPISINYRT LEGEND Alpha Helix
H Beta Sheet E Random Coil C
11
Structure
12
Motifs

• 1N-glycoslyation site
• 6 Protein kinase C phosphorylation
• 9 Casein kinase II phosphorylation

13
Motifs Contd

• 7 N-myristoylation site
• 1 Aldehyde dehydrogenases glutamic acid
• 1 Cell attachment site

14
N-Glycosylation Site

• Consensus sequence
• N-X-S/T/C
• Presence of Proline

15
Protein kinase C Phosphorylation
16
Casein kinase II Phosphorylation
17
N-Myristoylation Site
18
Aldehyde Dehydrogenase Glutamic Acid Active Site

• Responsible for oxidizing aliphatic or aromatic
  aldehyde
• Consensus sequence GETGGNSPV
• E is the active site

19
Cell Attachment Site

• Consensus Sequence RGD
• Plays a role in cell adhesion

20
Rooted Phylogenetic Tree
Silurus asotus
www.planetcatfish.com/.../silurus/asotus/7.jpg
Danio rerio
www.fishbase.org/images/species/Darer_m0.jpg
Homo sapiens
www.kidspast.com/images/homo-sapien-sapien.jpg
Mus musculus
www.upload.wikimedia.org/wikipedia/commons/6/62
/Mus_Musculus-huismuis.jpg
Gallus gallus
www.dkimages.com/discover/previews/905/670951.JPG
www.repti.net/.../Pleurodeleswaltl_55765.jpg
Xenopus laevis
www.pollywog.co.uk/Xenopus_laevis.jpg
21
Species from Blast P
22
Clustal W Bootstrap Tree
Rooted Tree
)
Bootstrap values
23
Multiple Sequence Alignment
24
MSA (Box Shade)
Human Fibronectin demonstrates more than 80
identity with other mammalian species
Major cell adhesive sites including Arg-Gly-Asp
(RGD) are present in all of the species except
chicken
The position of proline, leucine, and tyrosine
are highly conserved, the roles of these amino
acids are not known

• Key
• Conserved Green
• Identical Yellow
• Similar Blue
• Different Red

Tryptophan, a non-polar amino acid, is an
important factor in forming electrostatic
interaction with ligand-receptor connection, this
could possibly form a direct binding site for
unknown ligand.
Type III module does not have any cysteine
residues present, and hence no disulfide bridges
25
PROTPARS Parsimonious Tree
Newick Format
26
RGD Alignment Sequence
RGD found in humans
RGD found in Mouse
27
RGD in Newt (P. waltlii)
RGD found in humans
28
Cn3D Alignment

• Redfowl Chicken
• N-Myristolyation Site
• 174 aligned residues
• RGD lacks

29
Functional Structural Similarities

• Important in regulating cell growth
• Chain A in Zebrafish shares 55 identity with the
  mouse, humans and rats fibronectin.
• Has additional LDV binding site
• The structural homology of catfish to the amino
  acid sequence in the protein in Zebrafish,
  African clawed frog, humans is 60, 47.6, 48.5

30
Work Cited

• McCarthy, James, et. al. "Human Fibronectin
  Contains Distinct Adhesion-and Motility-promoting
  Domanis for Metastatic Melanoma Cells." Journal
  of Cell Biology 102(1986) 179-188.
• Mori, Tsukasa, Wataru Kawaguchi, Ikuei Hiraka,
  Youichi Kurata, Tadashi Saito, and Naoyuki
  Uchida. (2007)."Isolation and Identification of
  Major Plasma Fibronectin CDNA From Japanese
  Catfish Silurus Asotus." Comparitive Biochemistry
  and Physiology 146 53-59.
• Sun, Lu, Zhiying Zou, Paul Collodi, Fang Xu,
  Xiafeng Xu, and Qingshun Zhao. (2005).
  "Identification and Characterization of a Second
  Fibronectin Gene in Zebrafish." Matrix Biology
  24 69-77.

31
Questions
Fibronectin
Hitaa Gandhi, BarShaina Lamar, Hardik Modi