Abstract
The 16 kDa prolactin fragment arises from partial proteolysis of the native 23 kDa prolactin pituitary hormone. The mammary gland has been involved in this processing, although it has not been clarified whether it occurs in stroma or epithelial cells or extracellularly. Also, the processing enzyme has not been defined yet. Here we show that the incubation medium of stroma-deprived mammary acini from lactating rat contains an enzymatic activity able to cleave, in a temperature- and time-dependent fashion, the 23 kDa prolactin to generate a 16 kDa prolactin detectable under reducing conditions. This cleavage was not impaired in the presence of hirudin, a thrombin inhibitor, but strongly weakened in the presence of pepstatin A, a cathepsin D inhibitor. Cathepsin D immuno-depletion abolished the capability of acini-conditioned medium to cleave the 23 kDa prolactin. Brefeldin A treatment of acini, a condition that largely abolished the apical secretion of milk proteins, did not impair the secretion of the enzymatically active single chain of cathepsin D. These results show that mature cathepsin D from endosomes or lysosomes is released, likely at the baso-lateral site of mammary epithelial cells, and that a cathepsin D-dependent activity is required to effect, under physiological conditions, the cleavage of 23 kDa prolactin in the extracellular medium. This is the first report demonstrating that cathepsin D can perform a limited proteolysis of a substrate at physiological pH outside the cell.
Highlights
Brefeldin A treatment of acini, a condition that largely abolished the apical secretion of milk proteins, did not impair the secretion of the enzymatically active single chain of cathepsin D. These results show that mature cathepsin D from endosomes or lysosomes is released, likely at the baso-lateral site of mammary epithelial cells, and that a cathepsin D-dependent activity is required to effect, under physiological conditions, the cleavage of 23 kDa prolactin in the extracellular medium
PRL exists in the pituitary and in serum in several molecular forms, some of them arising from alternative splicing of the PRL mRNA, other forms arising from post-translational processing of the full-length 23 kDa PRL, such as glycosylation, phosphorylation, polymerisation and proteolytic cleavage (Sinha, 1995)
This hour at 37°C was detectable as a single 23 kDa band (Fig. 1B, kDa rPRL we considered the possibility that a proteolytic enzyme lane 1)
Summary
Prolactin (PRL), an hormone with a wide variety of biological activities in the regulation of reproduction, osmoregulation and immunomodulation (Ben Jonathan et al, 1996; Bole-Feysot et al, 1998; Freeman et al, 2000; Goffin et al, 2002), exerts important effects on the function of the mammary gland including effects on the expression of a number of genes and on the secretion of lipids and milk proteins (Hennighausen et al, 1997; Ollivier-Bousquet, 1998).PRL exists in the pituitary and in serum in several molecular forms, some of them arising from alternative splicing of the PRL mRNA, other forms arising from post-translational processing of the full-length 23 kDa PRL, such as glycosylation, phosphorylation, polymerisation and proteolytic cleavage (Sinha, 1995). In lactating mammary tissue and in milk, in various animal species, the 23 kDa form, the 25 kDa glycosylated form and a number of smaller molecular forms of PRL have been detected (Sinha, 1995; Ellis and Picciano, 1995; Lkhider et al, 1996). Whether and how this molecular heterogeneity reflects the functional diversity of PRL effects remain largely obscure (Corbacho et al, 2002). These data underline the interest in defining the tissue and the metabolic pathways responsible for the generation of the 16 kDa PRL
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