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Fig. 4. Impaired chondroitin sulfation and altered aggrecan metabolism in
Jaws-/- limbs. (A) CS immunostaining in
hindlimb digits (E14.5). Higher-magnification views (top) reveal sparse
irregular staining in the Jaws-/- joint.
(B,C) Fluorophore-assisted carbohydrate electrophoresis analysis
of chondroitin sulfate (CS, B) and heparan sulfate (HS, C) in E14.5 limbs. The
relative proportions and percentages of disaccharides generated with the
glycosaminoglycan-specific lysases are shown. For HS, no disaccharides
containing 2-sulfated uronic acid (UA) or N,6-sulfated GlcN were detected.
galNAc, N-acetylgalactosamine; C0S, 
UA-galNAc; C4S, UA-galNAc4S;
C6S, UA-galNAc6S; glcNAc, N-acetylglucosamine; GlcN, UA-glcNAc;
GlcNS, UA-glcNS; GlcN(6S), UA-glcNAc6S.
*P<0.025; 
P<0.0001,
Jaws-/- versus wild type percentage (n=5 pairs
per genotype). (D) Comparable amounts (mean and s.d.) of total
glycosaminoglycans in wild type and Jaws-/- hindlimbs,
normalized to wet tissue weight. Similar results were obtained for forelimbs.
n.d., not determined; HA, hyaluronan. (E) Aggrecan immunostaining in
the proximal end of the humerus (E14.5). (F) Anti-CDAGWL immunoblots of
E14.5 limb protein extracts, showing less full-length aggrecan (arrow,
350 kDa) and fewer aggrecan cleavage fragments (bracket) in
Jaws-/- cartilage. Successive nondissociative extractions
(lanes 1 and 2; arrowhead indicates a 65 kDa proteolytic fragment) were
followed by one dissociative extraction (lane 3). HL, hindlimbs; FL,
forelimbs.
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