Processing of the gap junction protein connexin50 in the ocular lens is accomplished by calpain.
Gap junction channel forming connexins share a common membrane topology which has four transmembrane spanning segments with the amino- and carboxy termini both located on the cytoplasmic side. Both, mutation and truncation of the carboxyl tail of some connexins have been shown previously to have profound effects on channel function. Truncation of the carboxyl tail of connexin50 (Cx50) and connexin46 (Cx46) occurs naturally during the maturation of fiber cells in the mammalian lens. This system therefore offers the unique opportunity to study not only the cleavage process but also the functional role played by the cleaved domain, in a physiologically relevant context. As a first step, we now report on the cleavage of the 70 kDa ovine isoform of Cx50. The calcium-activated neutral protease calpain (EC 3.4.22.17) was identified as the enzyme which removed a 32 kDa carboxyl portion from the Cx50 molecule in mature lens fiber cells. The amino-terminal 38 kDa portion remained embedded in the plasma membrane and was isolated and visualized as channel structures. The amino-terminal sequence of the cleaved 32 kDa portion matched an interior portion of the published amino acid sequence of the ovine Cx50 isoform. Thus, two closely spaced calpain cleavage sites were identified in the Cx50 molecule which were located carboxy-terminal from the predicted exit of the fourth transmembrane spanning segment by 62 or 72 amino acid residues, respectively. These data provide the basic information required for the future construction of Cx50 mutants to explore the functional consequences of this cleavage.
Lin
J S
JS
School of Biological Sciences, University of Auckland, New Zealand.
Fitzgerald
S
S
Dong
Y
Y
Knight
C
C
Donaldson
P
P
Kistler
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002154:Calpain / Q000378:metabolism*; D017630:Connexins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005136:Eye Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D007908:Lens, Crystalline / Q000378:metabolism* / Q000648:ultrastructure; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012756:Sheep; D012995:Solubility; D000099264:Connexin 50
D002154:Calpain; D017630:Connexins; D005136:Eye Proteins; D010446:Peptide Fragments; D000099264:Connexin 50