Overexpression of uteroferrin, a lysosomal acid phosphatase found in porcine uterine secretions, results in its high rate of secretion from transfected fibroblasts.
Lysosomal enzymes can, under certain circumstances, be secreted in large amounts. One example is uteroferrin (Uf), an iron-containing, purple-colored acid phosphatase secreted by the uterus of the pig during pregnancy. Uf is identical to the intracellular tartrate-resistant acid phosphatase of pig spleen, yet is the major protein component of uterine secretions. To investigate possible regulatory mechanisms that might direct Uf along a secretory pathway, we expressed Uf in Chinese hamster ovary (CHO) cells under the control of the SV40 early promoter using an expression construct, pX/Uf. The proportion of Uf secreted into the medium relative to the amount retained intracellularly increased as total Uf expression was increased. At transfection doses of 15 micrograms pX/Uf per 10(6) cells, over 80% of the Uf produced in 48 h was secreted. A parallel situation was observed when human cathepsin D was overexpressed in CHO cells. Thus, high production of Uf, as occurs in the uterus in response to progesterone, may overwhelm the intracellular enzymatic and receptor systems that are normally employed to target acid hydrolases to lysosomes, resulting in secretion. Both Uf and cathepsin D secreted by CHO cells possess N-linked, phosphorylated high-mannose oligosaccharide chains. However, the phosphate groups on the oligosaccharide chains of Uf, unlike those on cathepsin D, cannot be readily removed by alkaline phosphatase treatment. These results suggest that the phosphate groups on Uf are masked at least partially by covering N-acetylglucosamine residues and that two mechanisms may contribute to hypersecretion of Uf in the uterus: 1) very high rates of synthesis and 2) partial masking of the mannose 6-phosphate recognition signal.
Ling
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P
Department of Biochemistry, University of Missouri-Columbia 65211.
Roberts
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RM
D000135:Acid Phosphatase; D000818:Animals; D016466:CHO Cells; D002402:Cathepsin D / Q000235:genetics / Q000378:metabolism; D006224:Cricetinae; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006031:Glycosylation; D007163:Immunosorbent Techniques; D007527:Isoenzymes; D008247:Lysosomes / Q000201:enzymology*; D008667:Metalloproteins / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D013552:Swine / Q000378:metabolism*; D000071681:Tartrate-Resistant Acid Phosphatase; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology; D014599:Uterus / Q000201:enzymology* / Q000378:metabolism
D007527:Isoenzymes; D008667:Metalloproteins; D014415:Tunicamycin; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase; D002402:Cathepsin D