Aljj. Bronckers et al., DEGRADATION OF HAMSTER AMELOGENINS DURING SECRETORY STAGE ENAMEL FORMATION IN ORGAN-CULTURE, Matrix biology, 14(7), 1995, pp. 533-541
Increasing amelogenin heterogeneity during pre-eruptive enamel formati
on has been explained by proteolytic cleavage of a parent amelogenin,
differences in posttranslational modicifactions, translation of multip
le alternative spliced mRNA transcripts or combinations of these possi
bilities. We investigated the possibility of proteolytic degradation o
f amelogenins during secretory amelogenesis by pulse-labelling ameloge
nins with [H-3]proline followed by a pulse chase, all under organ cult
ure conditions. The results indicate that during pulse chase, hamster
molar tooth explants rapidly released substantial amounts of the radio
activity into the culture medium, as non-trichloroacetic-acid precipit
able, noncollagenous H-3-activity at the expense of radioactivity asso
ciated with the proteins in the enamel space. Simultaneously, there wa
s a continuous mineralization of the forming enamel in vitro as shown
by an increase in total calcium content of the explants. Western blott
ing, microdissection studies and fluorography of radiolabelled matrix
proteins after SDS-PAGE indicated that after an 8-h labelling, three r
adioactive amelogenin species could be extracted from forming enamel,
one prominent species of molecular mass 26 kDa and two less prominent
ones of 28 and 22 kDa. During pulse chase more amelogenin bands with l
ower molecular mass became apparent, a pattern similar to that observe
d in vivo. Examination of amelogenin blots with the glycan assay showe
d that none of the hamster amelogenins stained for carbohydrate. We co
nclude that changes in the amelogenin profiles during enamel developme
nt of cultured hamster explants are similar to those observed in vivo.
Although the formation of differently sized amelogenins from alternat
ively spliced mRNA transcripts cannot be ruled out, the in vitro pulse
-chase data obtained in this study suggest that amelogenin degradation
per se is probably responsible for most of the changes seen in the am
elogenin profiles.