Molecular Biology of the Extracellular Matrix 
Jeffrey p. Bonadio, M.D. — Assistant Investigator 
Dr. Bonadio is also Assistant Professor of Pathology and Member of the Program in Bioengineering at 
the University of Michigan Medical School. He received his bachelor's degree in biology from Marquette 
University and his M.D. degree from the Medical College of Wisconsin, Milwaukee. He studied anatomical 
pathology with Bruce Beckwith and medical molecular genetics with Peter Byers at the University 
of Washington, Seattle. 
THE long-term goal of our research is to under- 
stand how extracellular matrix proteins con- 
tribute to skeletal structure and function. Quanti- 
tative and qualitative changes in these proteins 
occur during morphogenesis and as part of the 
wound healing process. These observations sug- 
gest that both the organization and protein com- 
position of the matrix are precisely regulated. It 
is clear that this regulation occurs in part at the 
level of gene expression and in part at the level of 
the assembly of proteins into a matrix-like 
configuration. 
I have chosen to focus for the most part on the 
matrix molecule type I collagen. This collagen is 
a polymer of two related proteins whose se- 
quence has been determined. Moreover, the mul- 
tidomain structure of the molecule and a general 
outline of collagen biosynthesis are known, and 
the molecule is recognized to be distributed 
widely within tissues such as bone, tendon, liga- 
ment, tooth, dermis, and sclera. Previous studies 
have implied that type I collagen makes an im- 
portant contribution to the structure, integrity, 
and normal homeostasis of these tissues. Over the 
past year we have continued our work to establish 
model systems that would allow us to study this 
contribution at the molecular level. 
One system is designed to investigate the intra- 
cellular assembly of the collagen molecule. In 
general, this work involves site-specific mutagen- 
esis and assays that quantify the effects of muta- 
tion on the assembly process. These effects are 
studied at two levels. First, we have established 
conditions that allow synthetic peptides to fold 
into a collagen-like triple helix. Peptide folding 
is slow enough that the process can be character- 
ized by methods such as circular dichroism. In 
addition, the triple helix formed in vitro is stable 
enough that its structure can be characterized by 
nuclear magnetic resonance (NMR) techniques. 
Therefore the effect of a given mutation can be 
quantified by directly comparing the behavior of 
a normal peptide with that of mutant peptide. 
Second, cellular transfection methods have been 
developed to express and assemble collagen mol- 
ecules in vitro. Again, the effect of mutation on 
the assembly process can be quantified by di- 
rectly comparing the behavior of normal and mu- 
tant molecules. 
In our initial mutagenesis experiments, we 
characterized a highly conserved region of the 
triple-helical domain and demonstrated that it 
made an important contribution to the assembly 
of collagen molecules into a thermodynamically 
stable conformation. We speculate that this re- 
gion was conserved during evolution because it 
plays an important role in collagen biosynthesis, 
i.e., in folding the collagen molecule into its 
correct conformation. In the future, we hope to 
use this model system to define further the nor- 
mal contribution made by other collagen do- 
mains to the assembly process. In addition, we 
are interested in characterizing those regions of 
the molecule that mediate interactions between 
collagen and other matrix molecules such as 
fibronectin, heparin sulfate proteoglycan, and in- 
tegrins. These interactions are important because 
they represent a molecular basis for the assembly 
of collagen within the matrix. 
A second system is designed to investigate the 
function of type I collagen at the level of connec- 
tive tissue. Our initial set of experiments utilized 
a transgenic mouse strain that expressed only half 
the normal amount of type I collagen. We demon- 
strated that the mutation adversely affected the 
connective tissue of bone and skin dermis. In ad- 
dition, the mutant mice were profoundly deaf. 
We utilized biomechanical tests to quantify the 
effect of the collagen deficiency at the tissue 
level, and these studies demonstrated that the 
major role of type I collagen is to provide con- 
nective tissue with a high degree of resiliency. 
More recently, we also demonstrated that the 
skeleton of these transgenic mice is able to adapt 
to the inherited collagen deficiency. This adapta- 
tion involves a thickening of cortical bone and 
results from the synthesis of new bone matrix. 
Particularly intriguing was our observation that 
the adaptation was associated with a significant 
improvement in bone strength. This result is im- 
portant because it suggests the basis for a strategy 
to strengthen the fragile skeleton. 
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