Complete sequence analysis of cDNA clones encoding rat whey phosphoprotein: homology to a protease inhibitor.

Abstract

Lactoprotein clones have been isolated from a rat mammary gland recombinant library of cDNA plasmids. Clones p-Wp 52 and p-Wp 47 were shown by hybrid selection, in vitro translation, and immunoprecipitation to represent a cloned DNA sequence encoding rat whey phosphoprotein. We report here the nucleotide sequence of the cDNA insert ofp-Wp 52 and show that it encodes the complete whey phosphoprotein sequence. The encoded sequence shows a high content ofhalf-cystine, glutamic acid, aspartic acid, and serine but an absence of tyrosine. The half-cystines appear in unique arrangements and are repeated in two domains of the protein. The second domain has striking similarities with the second domain of the red sea turtle protease inhibitor. Clone p-Wp 52 has allowed the study of expression of whey phosphoprotein mRNA during functional differentiation of rat mammary gland and in mammary tumors. The whey phosphoprotein mRNA is detected during midpregnancy and lactation in the rat mammary gland but is barely detected in mammary tumors in which other milk protein mRNAs are expressed. The whey phosphoprotein gene in these tumors is hypermethylated, correlating with the reduced expression of this gene.Hormonal regulation of milk protein synthesis is being studied in a number of laboratories. From rat milk, a-lactalbumin (a-LA) (1, 2) and a number of caseins (42, 29, and 25 kilodaltons) (3, 4) have been purified and characterized. A unique group of phosphoproteins, electrophoretically distinct but having similar amino acid compositions, has been isolated from rat milk whey (1,5,6). This group ofwhey phosphoproteins (Wp-protein) constitutes 1 to 2 mg/ml in whole rat milk. These Wp-proteins initially copurify with rat a-LA in the whey fraction, but they can be separated from a-LA on DEAE-cellulose into three charged forms (1, 5, 6). These proteins are rich in aspartic and glutamic acids (or their amides) and in serine and half-cystine but lack tyrosine (5). The role ofthese proteins in the rat mammary gland is at present unknown, but their expression shows variability during functional differentiation of the gland.cDNA clones corresponding to some of the rat milk proteins have been isolated (7,8). For studies on the regulation of individual milk proteins, which are under different hormonal controls (9), it is essential that the individual cDNA clone be correctly identified with respect to its corresponding protein.Previously, we reported the isolation of a rat a-LA cDNA clone and established its identity by comparing the DNA sequence with the a-LA protein sequence (7).From taining a-LA also contained Wp-proteins. These fractions were then loaded on a DEAE-cellulose column and the proteins were eluted as described (1, 5, 6). The three charged forms of the phosphoproteins that did not have a-LA activity eluted after the a-LA protein peaks and were designated as Wpl, Wp2, and

Contrôle hormonal du développement et de l'activité de la glande mammaire

“…Un progrès assez net a également été réalisé dans la connaissance de la structure et la régulation des gènes cibles de la prolactine : caséines, alactalbumine, WAP, /3-lactoglobuline (Blackburn et al, 1982 ;Dandekar et al, 1982 ;Gupta et al, 1982 ;Hennighausen et al, 1982a, b ;Horn et al, 1983 ;YuLee et Nakhasi et al, 1984 ; Safaya, 1984 ;Houdebine et a/., 1985b …”

Inhibitory Function of Whey Acidic Protein in the Cell-Cycle Progression of Mouse Mammary Epithelial Cells (EpH4/K6 Cells)

“…Expression of the WAP gene is regulated by lactogenic hormones, and the level of mo use WAP m RN A in the m am m ary gla nds incr eases t hous ands -fo ld between non-lactation and mid-lactation [10]. WAP proteins have a signal peptide [11] and two domain structures that are identified at the fourdisulfide core (4-DSC) domain, which is composed o f ei g h t c y s t ei n e r es id ue s i n a c o ns er v ed arrangement [12]. The 4-DSC domain arrangement is not exclusive to the WAP family, and a number of other proteins containing 4-DSC domains have been identified as protease inhibitors [11,[13][14][15][16], comprising either single or multiple copies of the 4-DSC domain.…”

The acid‐stable proteinase inhibitor of human mucous secretions (HUSI‐I, antileukoprotease)

“…It is this second domain that is homologous to the two domains of HUSI-I and, as was known before [16], to both domains of the whey proteins. Attempts to detect an inhibitory activity of rat whey protein have failed f16).…”