Secretion of recombinant human fibrinogen by the murine mammary gland.
Transgenic animals secreting individual chains and assembled fibrinogen were produced to evaluate the capacity of the mammary gland for maximizing assembly, glycosylation and secretion of recombinant human fibrinogen (rhfib). Transgenes were constructed from the 4.1 kbp murine Whey Acidic Protein promoter (mWAP) and the three cDNAs coding for the Aalpha, Bbeta and gamma fibrinogen chains. Transgenic mice secreted fully assembled fibrinogen into milk at concentrations between 10 and 200 microg/ml, with total secretion of subunits approaching 700 microg/ml in milk. Partially purified fibrinogen was shown to form a visible and stable clot after treatment with human thrombin and factor XIII. The level of assembled fibrinogen was proportional to the lowest amount of subunit produced where both the Bbeta and gamma chains were rate limiting. Both the Bbeta and gamma chains were glycosylated when co-expressed and the degree of saccharide maturation was dependent on expression level, with processing preferred for gamma chains over Bbeta chains. Also, the subunit complexes gamma2, Aalphagamma2 and the individual subunits Aalpha, Bbeta and gamma were found as secretion products. When the Bbeta was secreted individually, the glycosylation profile of the molecule was of a mature complex saccharide indicating recognition of the molecule by the glycosylation pathway without association with other fibrinogen chains. To date secretion of Bbeta chain has been not observed in any cell type, suggesting that the secretion pathway in mammary epithelia is less restrictive than that occurring in hepatocytes and other cells previously used to study fibrinogen assembly.
Butler
Stephen P
SP
Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, USA.
O'Sickey
Tanya K
TK
Lord
Susan T
ST
Lubon
Henryk
H
Gwazdauskas
Francis C
FC
Velander
William H
WH
D000818:Animals; D005176:Factor XIII / Q000378:metabolism*; D005260:Female; D005340:Fibrinogen / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007774:Lactation; D008321:Mammary Glands, Animal / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D008892:Milk / Q000378:metabolism*; D008894:Milk Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism
D008894:Milk Proteins; D011994:Recombinant Proteins; C024471:whey acidic proteins; D005340:Fibrinogen; D005176:Factor XIII; D013917:Thrombin