Human and monkey lenses cultured with calcium ionophore form alphaB-crystallin lacking the C-terminal lysine, a prominent feature of some human cataracts.
OBJECTIVE
Elevation of lens calcium occurs in both human and experimental animal cataracts, and opacification may result from calcium-activated proteolysis. The purpose of the present study was to determine whether calcium accumulation in cultured human and Macaca mulatta lenses results in proteolysis of crystallins, the major lens proteins.


METHODS
Two-dimensional electrophoresis and mass spectrometry were used to construct detailed maps of human and monkey lens crystallins so that proteolysis after calcium accumulation could be monitored and the altered crystallins identified. Human and macaque lenses cultured in A23187 showed elevated lenticular calcium and superficial cortical opacities. The carboxypeptidase E (CPE) gene is expressed in human lens, and its presence in lens fibers was demonstrated by Western blot. To investigate whether CPE could cause similar truncation, purified alphaB-crystallin and CPE were incubated in vitro.


RESULTS
The major change observed in the crystallins of these cultured lenses was the accumulation of alphaB(1-174)-crystallin resulting from the loss of a C-terminal lysine. This result was significant, because similar appearance of alphaB(1-174) is a prominent change in some human cataracts. alphaB-crystallin and CPE incubation result in the formation of alphaB(1-174)-crystallin. This truncation was specific to alphaB(1-174)-crystallin, since other crystallins were not proteolyzed. Although a weaker activator than zinc, calcium activated CPE in vitro.


CONCLUSIONS
Since zinc concentrations did not increase during culture in A23187, calcium uptake in the lens may be responsible for CPE activation and alphaB(1-174) formation during cataract.
Nakajima
Emi
E
Laboratory of Ocular Sciences, Senju Pharmaceutical Corporation Limited, Beaverton, Oregon 97006, USA. nakajima@ohsu.edu
David
Larry L
LL
Riviere
Michael A
MA
Azuma
Mitsuyoshi
M
Shearer
Thomas R
TR
D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D015153:Blotting, Western; D000001:Calcimycin / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D043423:Carboxypeptidase H / Q000235:genetics / Q000378:metabolism; D002386:Cataract / Q000378:metabolism*; D002675:Child, Preschool; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007476:Ionophores / Q000494:pharmacology*; D007908:Lens, Crystalline / Q000187:drug effects* / Q000378:metabolism; D008239:Lysine / Q000378:metabolism*; D008253:Macaca mulatta; D013058:Mass Spectrometry; D009924:Organ Culture Techniques; D015032:Zinc / Q000378:metabolism; D038203:alpha-Crystallin B Chain / Q000737:chemistry / Q000378:metabolism*
D007476:Ionophores; D038203:alpha-Crystallin B Chain; D000001:Calcimycin; D043423:Carboxypeptidase H; D015032:Zinc; D008239:Lysine; D002118:Calcium