[Frontiers in Bioscience 2, a1-8, May 1, 1997]

	[Frontiers in Bioscience 2, a1-8, May 1, 1997] Reprints PubMed CAVEAT LECTOR
	THE CARBOXY-TERMINAL DOMAIN OF HUMAN SURFACTANT PROTEIN B IS NOT REQUIRED FOR SECRETION IN MILK OF TRANSGENIC MICE Sinai Yarus¹, Timothy E. Weaver², and Jeffrey M. Rosen¹ ¹ Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030-3498, USA ² Division of Neonatology and Pulmonary Biology, University of Cincinnati College of Medicine and Children's Hospital Medical Center, Cincinnati, Ohio 45267-0541, USA Received 3/24/97; Accepted 4/22/97; On-line 5/1/97 5. DISCUSSION The expression of the WAP/SP-BDELTAC genomic construct in the mammary gland of transgenic mice has been demonstrated, and the expected SP-BDELTAC 28 kDa protein was secreted in the milk. This confirms the earlier observations in cell culture (2) and in transgenic mice (15) which suggested that the carboxy-terminal domain of SP-B is not required for secretion. The ability to produce partially processed hSP-B in milk should facilitate the attainment of the long term goal of this project, i.e. the production of fully processed SP-B for pharmaceutical use. These and the previously published results (1) suggest that the enzyme necessary for cleavage of the amino-terminal propeptide of SP-B is not available in the mammary gland, even in the absence of possible steric hindrance from the carboxy-terminal propeptide. In a recent report, SP-BDELTAC targeted to the lung was shown to be processed to an active molecule in that organ (15), ruling out the possibility that the carboxy-terminal domain is required for the correct folding of the active SP-B molecule. Since the enzyme responsible for the cleavage of the amino-terminal domain of SP-B remains unidentified, the next step toward production of the fully processed molecule will be insertion of a chemical or enzymatic cleavage site (via site directed mutagenesis) between the amino-terminal domain and the mature peptide domain (16). One transgenic line expressing SP-BDELTAC produced an aberrant mammary gland phenotype characterized by decreased lobulo-alveolar development and led to the retardation of growth in the suckling neonates. Since little is known about the physical-chemical properties of SP-BDELTAC, its mechanism of action in producing the observed phenotype is unclear. No morphologic abnormalities result when SP-BDELTAC is targeted to the lung (17), but in that organ the SP-BDELTAC molecule is processed to mature SP-B. Similar phenotypes have been observed previously in both mice and swine overexpressing mouse WAP (18, 19), as well as transgenic mice expressing TGFbeta1 and a mutant p53 gene (20-22). Transplantation studies were used to show that the observed phenotype in the WAP/TGFbeta mice was due to a local effect of the transgene on the mammary epithelium (20, 21). The p53^arg-leu transgenic mice apparently suffer from decreased lobulo-alveolar development because of increased apoptosis (22), which is consistent with p53's role as a tumor suppressor gene. Since the observed phenotype in the SP-BDELTAC occurred in heterozygotes, insertional mutagenesis seems unlikely. The SP-BDELTAC protein apparently exerts its effect during pregnancy, since the aberrant morphology is apparent at day one of lactation. This observation is supported by previous work using the same WAP vector employed in this study (6, 7, 9, 1, 8) in which low level expression of heterologous transgenes in midpregnancy, a critical period for lobulo-alveolar development, may result in impaired lactation. This phenotype has not been observed with all WAP-based transgenes, or even for all lines generated from a single transgene construct. This implies the observed developmental abnormalities are the result of the specific properties of the overexpressed proteins, and their concentrations, rather than transcriptional or translational interference by the transgene. It is interesting that such diverse transgene proteins result in similar phenotypes. The mammary gland is apparently quite sensitive to disruptions in development during mid-pregnancy. The phenotype reported in this study is of interest primarily because it suggests that there may be an upper limit to the level of SP-BDELTAC which can be produced in milk. Hopefully, this will not impede development of production of SP-B for pharmaceutical use from milk.

[Frontiers in Bioscience 2, a1-8, May 1, 1997]
Reprints
PubMed
CAVEAT LECTOR

THE CARBOXY-TERMINAL DOMAIN OF HUMAN SURFACTANT PROTEIN B IS NOT REQUIRED FOR SECRETION IN MILK OF TRANSGENIC MICE

Sinai Yarus¹, Timothy E. Weaver², and Jeffrey M. Rosen¹

¹ Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030-3498, USA

² Division of Neonatology and Pulmonary Biology, University of Cincinnati College of Medicine and Children's Hospital Medical Center, Cincinnati, Ohio 45267-0541, USA

Received 3/24/97; Accepted 4/22/97; On-line 5/1/97

5. DISCUSSION

The expression of the WAP/SP-BDELTAC genomic construct in the mammary gland of transgenic mice has been demonstrated, and the expected SP-BDELTAC 28 kDa protein was secreted in the milk. This confirms the earlier observations in cell culture (2) and in transgenic mice (15) which suggested that the carboxy-terminal domain of SP-B is not required for secretion. The ability to produce partially processed hSP-B in milk should facilitate the attainment of the long term goal of this project, i.e. the production of fully processed SP-B for pharmaceutical use. These and the previously published results (1) suggest that the enzyme necessary for cleavage of the amino-terminal propeptide of SP-B is not available in the mammary gland, even in the absence of possible steric hindrance from the carboxy-terminal propeptide. In a recent report, SP-BDELTAC targeted to the lung was shown to be processed to an active molecule in that organ (15), ruling out the possibility that the carboxy-terminal domain is required for the correct folding of the active SP-B molecule. Since the enzyme responsible for the cleavage of the amino-terminal domain of SP-B remains unidentified, the next step toward production of the fully processed molecule will be insertion of a chemical or enzymatic cleavage site (via site directed mutagenesis) between the amino-terminal domain and the mature peptide domain (16).

One transgenic line expressing SP-BDELTAC produced an aberrant mammary gland phenotype characterized by decreased lobulo-alveolar development and led to the retardation of growth in the suckling neonates. Since little is known about the physical-chemical properties of SP-BDELTAC, its mechanism of action in producing the observed phenotype is unclear. No morphologic abnormalities result when SP-BDELTAC is targeted to the lung (17), but in that organ the SP-BDELTAC molecule is processed to mature SP-B. Similar phenotypes have been observed previously in both mice and swine overexpressing mouse WAP (18, 19), as well as transgenic mice expressing TGFbeta1 and a mutant p53 gene (20-22). Transplantation studies were used to show that the observed phenotype in the WAP/TGFbeta mice was due to a local effect of the transgene on the mammary epithelium (20, 21). The p53^arg-leu transgenic mice apparently suffer from decreased lobulo-alveolar development because of increased apoptosis (22), which is consistent with p53's role as a tumor suppressor gene. Since the observed phenotype in the SP-BDELTAC occurred in heterozygotes, insertional mutagenesis seems unlikely. The SP-BDELTAC protein apparently exerts its effect during pregnancy, since the aberrant morphology is apparent at day one of lactation. This observation is supported by previous work using the same WAP vector employed in this study (6, 7, 9, 1, 8) in which low level expression of heterologous transgenes in midpregnancy, a critical period for lobulo-alveolar development, may result in impaired lactation. This phenotype has not been observed with all WAP-based transgenes, or even for all lines generated from a single transgene construct. This implies the observed developmental abnormalities are the result of the specific properties of the overexpressed proteins, and their concentrations, rather than transcriptional or translational interference by the transgene. It is interesting that such diverse transgene proteins result in similar phenotypes. The mammary gland is apparently quite sensitive to disruptions in development during mid-pregnancy.

The phenotype reported in this study is of interest primarily because it suggests that there may be an upper limit to the level of SP-BDELTAC which can be produced in milk. Hopefully, this will not impede development of production of SP-B for pharmaceutical use from milk.