High expression of the human hepatocarcinoma-intestine-pancreas/pancreatic-associated protein (HIP/PAP) gene in the mammary gland of lactating transgenic mice. Secretion into the milk and purification of the HIP/PAP lectin.
The human hepatocarcinoma-intestine-pancreas/pancreatic-associated protein (HIP/PAP) gene was previously identified because of its increased expression in primary liver cancers and during the acute phase of pancreatitis. In normal tissues, HIP/PAP is expressed both in endocrine and exocrine cells of the intestine and pancreas. HIP/PAP is a lactose binding C-type lectin which acts as an adhesion molecule for rat hepatocytes. The aim of the work was to study the HIP/PAP secretory pathway and to produce high levels of HIP/PAP in the milk of lactating transgenic mice. In view of its lactose C-type lectin properties, we have studied the consequences of the expression of HIP/PAP on mammary epithelial cells. In homozygous mice, production reached 11.2 mg.mL-1 of milk. High levels of soluble and pure HIP/PAP (18.6 mg) were purified from 29 mL of milk. The purified protein was sequenced and the N-terminal amino acid of the mature HIP/PAP was identified as Glu27, thus localizing the site of cleavage of the signal peptide. The HIP/PAP transgene was only expressed in the mammary gland of lactating transgenic mice. HIP/PAP was detected by immunofluorescence in the whole gland, but labelling was heterogeneous between alveolar clusters, with strongly positive sparse cells. Using immuno electron microscopy, HIP/PAP was observed in all the compartments of the secretory pathway within the mammary epithelial cells. We provide evidence that HIP/PAP is secreted through the Golgi pathway. However, the number of distended Golgi saccules was increased when compared to that found in wild-type mouse mammary cells. These modifications could be related to HIP/PAP C-type lectin specific properties.
Christa
L
L
Institut National de la Santé et de la Recherche Médicale U-370 and Liver Unit, Centre Hospitalier Universitaire Necker, Paris, France. christa@necker.fr
Pauloin
A
A
Simon
M T
MT
Stinnakre
M G
MG
Fontaine
M L
ML
Delpal
S
S
Ollivier-Bousquet
M
M
Bréchot
C
C
Devinoy
E
E
D000209:Acute-Phase Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D002364:Caseins / Q000096:biosynthesis; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D007150:Immunohistochemistry; D007774:Lactation; D037181:Lectins, C-Type; D008321:Mammary Glands, Animal / Q000378:metabolism* / Q000648:ultrastructure; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D008892:Milk / Q000737:chemistry*; D008894:Milk Proteins / Q000235:genetics / Q000302:isolation & purification; D000073718:Pancreatitis-Associated Proteins; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000378:metabolism; D011506:Proteins; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification
D000209:Acute-Phase Proteins; D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D002364:Caseins; D037181:Lectins, C-Type; D008894:Milk Proteins; D000073718:Pancreatitis-Associated Proteins; D021382:Protein Sorting Signals; D011506:Proteins; C000612141:REG3A protein, human; D011993:Recombinant Fusion Proteins; C490253:Reg3b protein, mouse; C024471:whey acidic proteins