SURFACE LOCATED PROCOLLAGEN N-PROPEPTIDES ON DERMATOSPARACTIC COLLAGEN FIBRILS ARE NOT CLEAVED BY PROCOLLAGEN N-PROTEINASE AND DO NOT INHIBIT BINDING OF DECORIN TO THE FIBRIL SURFACE
Rb. Watson et al., SURFACE LOCATED PROCOLLAGEN N-PROPEPTIDES ON DERMATOSPARACTIC COLLAGEN FIBRILS ARE NOT CLEAVED BY PROCOLLAGEN N-PROTEINASE AND DO NOT INHIBIT BINDING OF DECORIN TO THE FIBRIL SURFACE, Journal of Molecular Biology, 278(1), 1998, pp. 195-204
Dermatosparaxis is a recessive disorder of animals (including man) whi
ch is caused by mutations in the gene for the enzyme procollagen N-pro
teinase and is characterised by extreme skin fragility. Partial loss o
f enzyme activity results in accumulation of pNcollagen (collagen with
N-propeptides) and abnormal collagen fibrils in the fragile skin. How
the N-propeptides persist in the tissue and how abnormal fibril morph
ology results in fragile skin is poorly understood. Using biochemical
and quantitative mass mapping electron microscopy we showed that the c
ollagen fibrils in the skin of a dermatosparactic calf contained 57% t
ype I pNcollagen and 43% type I collagen and the fibrils were irregula
rly arranged in bundles and hieroglyphic in cross-section. Image analy
sis of the fibril cross-sections suggested that the deviation from cir
cularity of dermatosparactic fibrils was caused by N-propeptides of pN
collagen being located at the fibril surface. Comparison of experiment
al and theoretical axial mass distributions of the fibrils showed that
the N-propeptides were located to the overlap zone of the fibril D-pe
riod (where D = 67 nm, the characteristic axial periodicity of collage
n fibrils). Treatment of the dermatosparactic fibrils with N-proteinas
e did not remove the N-propeptides from the fibrils, although the N-pr
opeptides were efficiently removed by trypsin and chymotrypsin. Howeve
r, the N-propeptides were efficiently cleaved by the N-proteinase when
the pNcollagen molecules were extracted from the fibrils. These resul
ts are consistent with close packing of N-propeptides at the fibril su
rface which prevented cleavage by the N-proteinase. Long-range axial m
ass determination along the fibril length showed gross non-uniformity
with multiple mass bulges. Of note is the skin fragility in dermatospa
raxis, and also the appearance of mass bulges along the fibril long ax
is symptomatic of the fragile skin of mice which lack decorin. Western
blot analysis showed that the dermatosparactic fibrils bound elevated
levels of the proteoglycan, compared with normal skin fibrils. The re
sults showed that N-propeptides can distort the morphology of fibrils,
that they do not inhibit binding of gap-associated macromolecules (su
ch as decorin) and that the normal mechanical properties of skin are s
trongly dependent on the close association of near-cylindrical fibrils
, thereby enabling maximal fibril-fibril interactions. (C) 1998 Academ
ic Press Limited.