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Studying Limb Dysplasia Using Bioinformatics

Author: Heather Grisco, Undergraduate Student, Animal Science, UIUC
Instructor: Dr. Sandra Rodriguez-Zas, UIUC

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Objectives

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We will be using the biology workbench to examine the effects that gdf-5 (growth dependent factor 5) has on limb abnormalities.Ý We will be focusing primarily on the abnormalities caused by the transforming growth factor, tgf, proteins and how they affect growth, specifically causing limb dysplasia.

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Overview of Limb Dysplasia

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The two specific types of limb dysplasia that both are affected by tgf are Grebeís dysplasia and Hunter-Thompson dysplasia.Ý Both are very similar.Ý Grebeís dysplasia is a rare form of short limbed dwarfism that can be inherited.ÝA person suffering from this type of dysplasia would have missing or greatly shortened limbs.Ý Of those shortened limbs, the hands and feet are most greatly affected.Ý In the hands carpal bones are most affected.Ý They are abnormal and fused, metacarpals may be absent, and the proximal and middle phalanges are absent.Ý Others who are carriers of this disease may have some mild skeletal abnormalities.ÝFor example, the first metacarpal and middle phalanges of fingers two and five are shortened.Ý In both cases things like intelligence, emotions, and puberty are unaffected by the disease.

ÝÝÝÝÝÝÝÝÝÝÝ This disorder is caused by abnormalities in the cdmp1 gene (gdf5 for mice) which result in a dominant negative effect.Ý The abnormal protein forms nonfunctional heterodimers with other bmps preventing their ability to secrete signaling molecules.

ÝÝÝÝÝÝÝÝÝÝÝ Hunter-Thompson dysplasia is similar to Grebeís dysplasia in many ways, but it affects regions that are more specific.Ý For example, in both the hands and feet suffer most severely, but in Hunter-Thompson dysplasia, the lower limbs are still more likely to be affected than the upper limbs.

ÝÝÝÝÝÝÝÝÝÝÝ This disorder is specifically caused by a homozygous mutation in the CDMP1 gene.ÝThis mutation results in a frameshift that disrupts the highly conserved 7-cysteine pattern.Ý This cysteine pattern normally determines the protein structure and function.Ý In this case, when it is disrupted, improper tgfs are made which is what results in Hunter-Thompson dysplasia.

More information on CDMP1

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Using the Biology Workbench

ÝÝÝÝÝÝÝÝÝÝÝ If possible, open a second web browser and open the biology workbench (http://workbench.sdsc.edu) in it.Ý The best way to proceed through the tutorial is to set up two windows next to eachother, but this is not necessary.Ý You can also printout the tutorial.

ÝÝÝÝÝÝÝÝÝÝÝ For first time users, you will need to set up an account.Ý To do this, just click on set-up a free account and enter a user name and password.Ý Once you have done this once, you will not have to do it again.

ÝÝÝÝÝÝÝÝÝÝÝ Once registered, enter the Biology Workbench and click on the ìProtein Toolsî button.Ý Highlight the NDJINN Multiple Database Search from the scrollbar menu.Ý This allows the user to find information on a topic of interest, using specific databases that may be useful.Ý After highlighting the search, click ìrunî.

ÝÝÝÝÝÝÝÝÝÝÝ In the new menu, check the boxes next to PIR, PDBFINDER, and SWISSPROT.ÝThese are your selected databases.ÝThe protein information resource (PIR) database is useful for finding resources and amino acid compositions for different proteins.Ý PDBFINDER is useful in finding sequences derived from PDB, DSSP, and HSSP databases.ÝSWISSPROT is a database in Switzerland that stores the amino acid sequences that make up different proteins.ÝAny other databases can be chosen, these three are the most common and most useful for this project.

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ÝÝÝÝÝÝÝÝÝÝÝ Type: 

ÝÝÝÝÝÝÝÝÝÝÝ ÝÝÝÝÝÝÝÝÝÝÝ Gdf5

in the query space above the command buttons.

Submit your query (click on the ìsearchî button).

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Clustal-W Sequence Alignments

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In the menu, you will see two results, one for humans one for mice.Ý Check both boxes and then click the ìImport Sequencesî button.ÝThis will bring you back to the screen that you originally started with.Ý Re-check the two boxes that say SWISSPROT gdf5: human and mouse, in preparation to align the sequences.

ÝÝÝÝÝÝÝÝÝÝÝ Align the sequences using the Clustal-W tool.Ý Clustal-W is a tool used to align the protein sequences that we have just imported.Ý This is accomplished by highlighting the Clustal-W program in the scroll menu and selecting ìRunî.Ý The next screen presents an assortment of settings for the Clustal-W program.Ý We will not change any of these settings, so scroll down the page and click ìsubmitî.

Human-Mouse Sequence Alignment

ÝÝÝÝÝÝÝÝÝÝÝ The sequences should now be aligned.Ý All of the blue ones with an asterisk are completely conserved, the green : shows conservation of strong groups and the dark blue . shows conservation of weak groups.Ý Examine these sequences and look for what areas are conserved.Ý Are there many similarities?Ý Do certain base pairings always result in nonconserved regions?

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Comparing the Sequences

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Another way to compare your already aligned sequences is by using the BOX SHADE tool.ÝOnce your sequences are aligned, click on the ì Import Sequencesî button.Ý This will send you back to the initial screen.Ý Check the box marked Clustal-W protein for the gdfs that we just found.Ý Now highlight the BOX SHADE option from the scroll menu and hit ìRunî.Ý Another assortment of settings will be presented.Ý As with the CLUSTAL-W screen, scroll down to the bottom and hit ìSubmitî.Ý The BOX SHADE version will give you the aligned sequences this time focusing on the groups of proteins.Ý It will produce a color-coded output of the protein sequences. ÝIf you need to go back a page and check your color settings please do.Ý This will be helpful in comparing amino acids.Ý There are different colors for amino acids fully conserved in all proteins, nearly conserved, and not conserved, but still has the similar structure and charge.

ÝÝÝÝÝÝÝÝÝÝÝ Examine the aligned sequences again.Ý How similar are the amino acid sequences?Ý Knowing that three amino acids code for one protein, how similar would the protein sequences be for each animal?

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Nucleic Tools

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We can also do a comparison using the nucleic tools.Ý To do this get back to the original screen and select the ìNucleic Toolsî button.Ý Select NDJINN Multidatabase search from the scroll menu and type in gdf5, just like before and click ìRunî.Ý This time select the GBVRT, GBROD, and GBPRI databases and click ìSearchî.Ý Five results will appear, select the three that contain information about gdf5 and click the ìImport Sequencesî button.ÝThis will bring you back to the main page where you can perform a CLUSTAL-W alignment or a BOX SHADE comparison according to the same procedures used above.Ý Ask yourself how the three sequences are similar and how they are different.Ý Are there two sequences that are closer to eachother than the other?

Nucleic Sequence Alignment

TGF-Beta

ÝÝÝÝÝÝÝÝÝÝÝ The above searches can also be done with Tgf-beta. (Type in tgfb instead of gdf5)ÝRemember thatÝ the gdf5 that we have used in the previous searches is a product of the tgfs.Ý They (growth differentiating factors) are the signaling molecules that elicit their response by binding to specific receptors.Ý If these are damaged, heterodimer formation will still occur, but it will be nonfunctional because the gdfs/signaling molecules will not be functional.Ý This is how the dysplasia occurs.

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Quaternary Structure

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By using a different program, we can view the quaternary structure of a Tgf-beta protein.ÝThe quaternary structure of proteins is the total overall structure.Ý It is made when all of the subunits, primarily, secondary, and tertiary, are in place.Ý We have already viewed the primary and secondary structures.Ý The primary structure is composed of the nucleotides. (A, C, G, T)Ý These make up the DNA sequence that contains the code to make the protein.Ý The secondary structure is the amino acid sequence that makes up the protein.Ý The tertiary structure is the 3-D structure of the protein that allows it to perform its functions.

In order to view the quaternary structure we will use the Rasmol application.Ý Rasmol is a molecular modeling program that was written by Roger Sayle.Ý Tutorials on using Rasmol can be found at http://www.umass.edu/microbio/rasmol/rastut.htm

To access the Rasmol program go to http://www.umass.edu/microbio/rasmol

You may need to download this program.Ý To do this click on Rasmol.Ý Scroll down until you see the link ìgetting and installing Rasmolî, click on that.Ý Chose which type of program you would like to download it to and then click on the specified link.Ý Chose where you would like to save the program. (Ex. Save under My Computer, highlight C: and click save the file name will automatically be rw32b2a.exe)ÝÝ 

ÝÝÝÝÝÝÝÝÝÝÝ Once your file is downloaded, you can now begin to search.Ý Go back to the Rasmol homepage and click on ìdownload a free PDB data file.îÝ Scroll down to the green box that says, ìFind, View Download Macromolecules from the Protein Data Bank with PDB Liteî, click on this.Ý From here select Israel.Ý (The US mode does not always work properly)Ý You are now ready to begin your search.

ÝÝÝÝÝÝÝÝÝÝÝ Once this is finished you should be able to type in your selections.Ý 

Type: Tgf-beta

A box will come up giving the results of the query.Ý If you scroll down slightly a box that says, ìRetrieve released data matching your queryî will appear.ÝClick on it.Ý The results retrieved will include many different beta factors (ex. Alpha, 1, 2, etc.)Ý We will only look at beta1 and beta2 subunits.Ý 

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ÝÝÝÝÝÝÝÝÝÝÝ First select the file called 1klc and select view/analyze/save macromolecule.ÝSelect save 1klc.pdb and click on express save for experts.Ý If this does not work properly follow the saving instructions for your computer type.ÝA save as screen should pop up.ÝSelect where you want to save your macromolecule.Ý (It is probably easiest to save it under the same file as the original Rasmol program.ÝFor example, we saved it under My Computer, C: above.)Ý Once you have found where you would like to save it, click save.Ý After a few seconds of downloading the file will be in place.

ÝÝÝÝÝÝÝÝÝÝÝ Now its time to view our molecule.Ý Open the Rasmol program by clicking on the rw32b2a.exe file folder.ÝA black screen should appear.Ý On this screen go to file, click open, and find the molecule you just saved. (1klc)

ÝÝÝÝÝÝÝÝÝÝÝ A wire frame structure of tgf-beta1 should appear.Ý Get a good look at this molecule.Ý Shown is a drawing of the protein using a wireframe for the amino acids in the protein.Ý There are also many other ways to view this structure.Ý Try a few of them (read the manual for more descriptions of the options).

ÝÝÝÝÝÝÝÝÝÝÝ You can also rotate the molecule.Ý To do this, just hold it with the cursor and start moving it around.Ý Try to look at all angles of the molecules.ÝAre there certain groups that are more likely to be found on the inside of the molecule?Ý The outside?

ÝÝÝÝÝÝÝÝÝÝÝ It is really hard to see, but if you display the molecule using labels you may be able to find the highly conserved cysteine area.Ý Remember that it is a frameshift that disrupts this region and changes the function of the protein.

ÝÝÝÝÝÝÝÝÝÝÝ Do to this mutation, the protein molecule can bond with another molecule forming a nonfunctional heterodimer.Ý This prevents proper signaling molecule excretion and causes limb dysplasia. 

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ÝÝÝÝÝÝÝÝÝÝÝ Try to Visualize

Lets try to visualize the formation of the nonfunctional heterodimer.Ý Begin a new Rasmol search.Ý (You may need to go back to the initial Rasmol website. www.umass.edu/microbio/rasmol and repeat the steps as before)Ý This time search for bmp.Ý After retrieving the batch of results, select 1bmp.pdp and save this molecule to the same place you saved 1klc.

ÝÝÝÝÝÝÝÝÝÝÝ Open the Rasmol window (black screen) select open, and find the 1bmp.pdb file, open it.Ý Look closely at this molecule.ÝIf you can, open a second Rasmol screen and open the 1klc.pdb file.Ý By viewing them side by side, try to visualize where they would bind together.ÝCan you visually form the heterodimer?ÝThese two combined is what produces the functioning unit.Ý If either of these molecules is mutated, they will not pair properly and will therefore produce a nonfunctional heterodimer.

Just for Fun

If youíd like, try opening some of the other tgf-beta molecules and compare them to the tgf-beta1 molecule that we viewed.ÝAre there many similarities?ÝDifferences?Ý How do you think they would bind to the BMP?

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ÝÝÝÝÝÝÝÝÝÝÝ Genetics

Limb dysplasia is a hereditary disease; it can be passed down from generation to generation.ÝThose affected must be homozygous recessive for the trait that is; they need to have inherited it from both their mother and their father.Ý This can happen if both parents are carriers for the gene (25% chance), if one parent has the disease and the other is a carrier (50% chance), or if both parents have the disease (100% chance).

Although limb dysplasia is a rare disease, it is best to know if it is your family lines.Ý This way you help prevent producing children with the disease.

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ÝÝÝÝÝÝÝÝÝÝÝ Summary

Although genetics plays a role in creating dysplasia so does mutation, specifically frameshift mutation.Ý The most important idea behind this is that the frameshift mutation disrupts the highly conserved cysteine pattern of the molecule.Ý This pattern is what determines the protein structure. ÝIf the structure is abnormal, this abnormal protein will form heterodimers with other BMPs preventing the proper secretion of signaling molecules.

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References:

http://peptide.ncsa.uiuc.edu/tutorials_current/Sickle_Cell_Anemia/SC2001/

Grim, Meg Sickle-Cell Anemia tutorial

Holder-Espinasse, Lyonnet, Manouvrier-Hanu, ìGenetics of limb anomalies in humansî, ÝÝÝTIG Oct. 99, volume 15, No.10

http://www.graylab.ac.uk/omd/index.html

Cancer-Webís online medical dictionary

Links:

Related Information:

http://rarediseases.info.nih.gov/ord/news-reports/FY97annual/NICHD.htm

Office of rare diseases website.Ý Important information on various limb dysplasias and other rare disease.