Biochemistry Collagen Synthesis Disorder Case study: Your patient is Jane, a 10-
ID: 186558 • Letter: B
Question
Biochemistry
Collagen Synthesis Disorder
Case study:
Your patient is Jane, a 10-year-old girl of short stature with hyperelastic skin which bruises easily, and hypermobile joints which are easily dislocated. Jane's mother said that her diet includes fruits and vegetables high in Vitamin C, so she does not have a vitamin C deficiency. She also has no signs of anemia and has normal iron levels.
You decide to refer Jane to a surgeon who will correct the looseness of Jane's joints. The surgeon agrees to take skin samples during the surgery so that you can perform an analysis of her fibroblasts. These cells produce connective tissue in the skin. Because skin, ligaments, and tendons have collagen as their major structural protein, you suspect that Jane is unable to synthesize collagen properly and you wish to test this hypothesis.
Jane's symptoms sound remarkably similar to a condition called dermatosparaxis (literally “torn skin") that has been observed in sheep and cattle. The disorder probably is due to improper collagen formation, you get a sample of collagen from a dermatosparactic sheep to use in your experiments. You are able to obtain fibroblasts from the samples of Jane's skin and the skin from the dermatosparactic sheep, as well as normal control fibroblasts capable of secreting normal collagen.
Collagen is synthesized in fibroblast cells in the skin. There are four main types of collagen, but you suspect that the defect is in Type I collagen synthesis, because Type I collagen is predominantly distributed in tendons, bone, skin, and ligaments. Type I collagen consists of three chains: two identical 1(I) chains and an 2(I) chain. The biosynthetic pathway is can be found in the module notes and is shown below (Figure 1).
You have extracted and solubilized the collagen from the fibroblast cell cultures of your patient Jane, a dermatosparactic sheep, and normal control for your experiments. The patient’s collagen is much easier to extract than the control and sheep collagen.
You have taken the cells and collagen produced from those cells to the lab for further analysis.
In the first experiment, you carry out SDS-PAGE analysis of the collagen extracted from a normal control, patient, and sheep fibroblasts. The results are shown in Figure 2.
Analyzing the results of the gel you determine that Jane has 2 normal 1(I) chains, since the band is the same size and intensity as that observed in the control. Comparing Jane's 2 chain to that of the control, you note that there is a band that is the same size as the control, but it is half the intensity. A second band is observed at higher molecular weight, corresponding to the molecular weight of the 2 band for the sheep. In the sheep sample, both the 1(I) and 2 migrate slower through the gel than control, indicating a higher molecular weight. Using your knowledge of the biosynthetic pathway for collagen (Figure 1), you determine that the higher molecular weight band for 2 likely results from a defect in cleavage of the 2 chain.
Following up on the SDS-PAGE results discussed above, you carry out a series of experiments outlined in Tables 1, 2 and 3. In the first set of experiments, the cultured fibroblasts are assayed for levels of key enzymes necessary for proper collagen synthesis. In the second set of experiments, an amino acid analysis of control and patient collagen is carried out. In the third set of experiments, exogenous normal enzymes are added to the collagen from the cultured fibroblast medium to see if these enzymes can correct the defect. The results are shown in the tables.
Table 1: Assays of enzyme activity:
Jane’s fibroblast enzyme activity
Dermatosparactic sheep fibroblast enzyme activity
Procollagen N-peptidase
Normal
Low
Procollagen C-peptidase
Normal
Normal
Prolyl hydroxylase
Normal
Normal
Lysyl hydroxylase
Normal
Normal
Table 2: Relative amino acid compositions:
Collagen extracted from Jane’s fibroblasts
Collagen extracted from dermatosparactic sheep
4-Hydroxyproline ( 4-Hyp)
Normal
Normal
3-Hydroxyproline (3-Hyp)
Normal
Normal
Hydroxylysine (Hyl)
Normal
Normal
Table 3: Incubation of collagen with exogenous enzyme from a normal source:
Collagen extracted from Jane’s fibroblasts
Collagen extracted from dermatosparactic sheep
Procollagen N-peptidase
No change
SDS-PAGE results show collagen is the same as the control after digestion.
Procollagen C-peptidase
No change
No change
Using the analysis of the gel and one or more of the tables and how the data helps to determine a diagnosis for Jane. Use the following questions to guide your discussion.
1. What is the significance of knowing that Jane had plenty of vitamin C in her diet and that her iron levels are normal? Why did the amount of Vitamin C in Jane’s diet fail to improve Jane’s symptoms?
2. What do you think is the cause of the size differences in Jane’s collagen, and the sheep’s collagen?
Jane’s fibroblast enzyme activity
Dermatosparactic sheep fibroblast enzyme activity
Procollagen N-peptidase
Normal
Low
Procollagen C-peptidase
Normal
Normal
Prolyl hydroxylase
Normal
Normal
Lysyl hydroxylase
Normal
Normal
Ribosome Chaperonins mRNA Procollagen assembly 1. Chaperonins assist folding 2. Hydroxylation 3. N-linked glycosylation 4. Self-assembly, formation Procollagen Propeptide OH OH OH OH OH OH of disulfide bonds OH OH OH OH OH OH Cis O 5. Conversion of proline peptide bonds from cis to trans configuration Endoplasmic reticulum Vesicular tubule cluster 6. Formation of a triple helix 7. Transport through Golgi apparatus Cytosol Golgi apparatus 8. Modification of N- and O-linked sugars Fibril/fiber assembly 9. Cleavage of propeptides. 10. Self-assembly of fibril 11. Secretion 12. Fiber assembly r Trop°collagen Fibripositor Extracellular space Collagen fibrils Collagen fiberExplanation / Answer
1. Amount of Vitamin C in Jane’s diet fail to improve Jane’s symptoms because the dermatosparaxis is occurs due to the problem in the collagen molecule where it is important in connective tissue of skin, ligaments, and tendons. So vitamin C has no effect here.
2. Procollagen N-peptidase activity is totally arrested in the dermatosparaxis sheep. Thats why it not able to cleave the procollagen, so the molecular weight is increased and the collagen not able to migrate faster in SDS page. And also due to extra peptide present in the N termianl of collagen it not able to function properly. But in janes alpha 1 Procollagen N-peptidase working good so it cleave the N terminal and it match with Control group.
But in alpha 2 the janes collagen is match with control but the intensity is very low and als we are seeing 2 bands here. There is cleavage of alpha 2 collagen is seen here. So it makes problem in janes health condition.
Related Questions
Navigate
Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.