Henryk Lubon

Inefficient processing of human protein C in the mouse mammary gland

Vitamin K-dependent plasma protein, human Protein C (HPC) has been expressed in transgenic mice, using a 4.2kb mouse whey acidic protein (WAP) promoter, 9.0 kb HPC gene and 0.4 kb 3′flanking sequences. Expression was mammary gland-specific and the recombinant human Protein C (rHPC) was detected in milk at concentrations of 0.1 to 0.7mg ml−1. SDS-PAGE revealed that the single, heavy and light chains of rHPC migrated with increased electrophoretic mobility, as compared to HPC. Enzymatic deglycosylation showed that these molecular weight disparities are in part due to differential glycosylation. The substantial increase observed in the amount of single chain protein, as well as the presence of the propeptide attached to 20–30% of rHPC, suggest that mouse mammary epithelial cells are not capable of efficient proteolytic processing of rHPC. TheKm of purified rHPC for the S-2366 synthetic substrate was similar to that of plasma-derived HPC, while the specific activity was about 42–77%. Amino acid sequence analyses and low anticoagulant activity of purified rHPC suggest that γ-carboxylation of rHPC is insufficient. These results show that proteolytic processing and γ-carboxylation can be limiting events in the overexpression of fully biologically active rHPC in the mouse mammary gland.

Transgenic pigs as bioreactors: a comparison of gamma-carboxylation of glutamic acid in recombinant human protein C and factor IX by the mammary gland

The mammary gland of transgenic livestock can be used as a bioreactor for producing complex therapeutic proteins. However, the capacity for making a given post-translational modification upon any given polypeptide is uncertain. For example, the efficiency of gamma-carboxylation of glutamic acid in the amino terminal regions of recombinant human protein C (rhPC) and recombinant human Factor IX (rhFIX) is different at similar expression levels. At an expression level of about 200 microg/ml in the milk of transgenic pigs, rhFIX is highly gamma-carboxylated as indicated by pro-coagulant activity and amino acid sequencing. However, only about 20-35% of rhPC has a native, gamma-carboxyglutamic acid-dependent conformation and anti-coagulant activity. Thus, this work provides an example of apparent differences in substrate specificity between two homologous proteins to the endogenous carboxylase of porcine mammary epithelium which leads to varying degrees of post-translational modification.

Transgenic animal bioreactors in biotechnology and production of blood proteins.

The regulatory elements of genes used to target the tissue-specific expression of heterologous human proteins have been studied in vitro and in transgenic mice. Hybrid genes exhibiting the desired performance have been introduced into large animals. Complex proteins like protein C, factor IX, factor VIII, fibrinogen and hemoglobin, in addition to simpler proteins like alpha 1-antitrypsin, antithrombin III, albumin and tissue plasminogen activator have been produced in transgenic livestock. The amount of functional protein secreted when the transgene is expressed at high levels may be limited by the required posttranslational modifications in host tissues. This can be overcome by engineering the transgenic bioreactor to express the appropriate modifying enzymes. Genetically engineered livestock are thus rapidly becoming a choice for the production of recombinant human blood proteins.

The Porcine Mammary Gland as a Bioreactor for Complex Proteinsa

The similar biological activity of rhPC and hPC indicates that porcine mammary gland can perform many of the processing reactions necessary for recombinant synthesis of complex human proteins and produce them at levels suitable for industrial bioreactor applications. The health of the transgenic pigs appeared unaffected by the expression of high levels of the heterologous protein. We suggest that one of the advantages of using the mammary gland as a bioreactor appears to be the high cell density relative to that of cell culture.

Interactions between cellular regulatory proteins and a unique sequence region in the human cytomegalovirus major immediate-early promoter

Transcription from the human cytomegalovirus major immediate-early promoter is dependent on host-cell regulatory proteins. The interactions between cellular nuclear proteins and a unique sequence located from nucleotide position -660 to -540 was investigated. The unique region presents a defined target for multiple distinct DNA-binding proteins which appear, in part, to have overlapping binding sites. A minimum of five sequence-specific DNA-binding activities that interact with sequences between -632 and -602, -602 and -557, -602 and -590, -563 and -540, and -602 and -582 were detected. Evidence is presented to suggest that the -632 to -602 site, a previously characterized nuclear factor 1 binding site, does not bind NF1 but strongly interacts with a distinct cellular factor. The binding of cellular proteins to the unique sequence region was shown to be important in directing transcription from the major immediate-early promoter.

Transgenic mice expressing recombinant human protein C exhibit defects in lactation and impaired mammary gland development.

To determine if the production of recombinant human protein C (rHPC) could be increased in milk, we created two lines of mice homozygous for the mouse whey acidic protein (WAP)/human protein C (HPC) transgene. Females of both lines had normal growth, activity and fertility, but failed to lactate normally and were unable to raise litters. Histological analyses of mammary glands from lactating homozygous females showed barely distended alveoli filled with dense-staining milk. Epithelial cells within these alveoli had distinct, centrally located nuclei and contained intracellular lipid droplets. Hemizygous animals derived from these lines were able to lactate and raised normal sized litters. Northern blot analysis showed that the 6.4 homozygous (6.4H) line expressed the transgene at higher levels then corresponding hemizygous (6.4) animals, but the 4.2 homozygous (4.2H) line expressed the transgene at lower levels than the 4.2 hemizygous line. The 6.4H line also had increased rHPC levels in the milk as revealed by western blot analysis. The 4.2H, 6.4, and 6.4H lines showed decreased and/or delayed expression of WAP, β-casein, and α-lactalbumin mRNAs compared to wild type animals during lactogenesis. The 4.2 line showed decreased mRNA expression for β-casein and α-lactalbumin, but normal or higher expression of WAP during lactogenesis. Elevated levels of some proteins were detected in the milk of transgenic mice. From these results, it is concluded that expression of rHPC induced a lactational phenotype that involves abnormal morphological, biochemical, and functional differentiation of mammary epithelial cells. However, the induction of this phenotype does not appear to be directly related to the level of rHPC mRNA expression, thus suggesting that the basis of this phenotype may involve secondary, rather than primary, effects of rHPC on mammary gland development.

Regulation of human protein C gene expression by the mouse WAP promoter.

A 4.1 kb mouse whey acidic protein (mWAP) promoter was cloned from a C57BL/6 cosmid library. The tissue-specific and developmental pattern of expression of a hybrid gene comprised of the mWAP promoter fragment and the human protein C (HPC) gene was analysed in transgenic mice. The corresponding RNA was detected mainly in the mammary gland, with ‘leakage’ of expression in the salivary gland and kidney. The developmental pattern of transgene expression differed from that of the endogenous WAP gene. In particular, recombinant HPC (rHPC) transcripts were detected earlier in pregnancy than WAP RNA, with no significant increase during lactation. This indicates that regulatory elements responsible for developmental regulation are located outside the 4.1 kb mWAP gene promoter fragment, or if present, may be subject to position effects. Precocious expression of the transgene did not compromise the health or nursing abilities of transgenic females. Expression of rHPC affected the appearance of the mammary alveoli and alveolar epithelial cells in lactating transgenic mice. The alveoli were less distended and alveolar epithelial cells appeared cuboidal with centrally positioned nuclei. We suggest that the inefficient intracellular processing of rHPC can alter the histological appearance of alveolar epithelial cells in the transgenic mammary gland.

Phenotypic and genotypic stability of multiple lines of transgenic pigs expressing recombinant human protein C

The genotypic and phenotypic stability of four lines of transgenic pigs expressing recombinant human protein C in milk was examined. Two lines were established with a construct consisting of a 2.6 kb mouse WAP promoter and a 9.4 kb human protein C genomic DNA. Two lines were established with another construct consisting of a 4.1 kb mouse WAP promoter and a 9.4 kb human protein C genomic DNA. Genotypic stability was measured by transgene copy number transmission. Outbred offspring having a single transgene integration locus were established from a founder having three independent, multicopy loci. Phenotypic stability over multiple lactations was defined by the combination of recombinant human protein C expression levels and the isoform signature of recombinant human protein C in western blots. Both cDNA and genomic human protein C transgenes gave similar ranges of expression levels of about 100--1800 μg ml−1. Within a given outbred lineage having a single loci for the cDNA transgene, the expression levels ranged between 100--400 μg ml−1. Western blots of reduced recombinant protein C revealed that single chain content was not dependent on expression level and was consistent within each transgenic line, but varied between transgenic lines. This suggests that native swine genetics may play a role in selection of production herds with optimal post-translational proteolytic processing capability. Although swine are not conventional dairy livestock, it is agreed that the short generation times, multiple offspring per litter, stable paternal transmission of the transgene, and milk production capabilities of swine offer distinct advantages over conventional dairy livestock for the establishment of a herd producing a therapeutic recombinant protein

Affinity purification of biologically active and inactive forms of recombinant human protein C produced in porcine mammary gland

Recombinant human protein C (rhPC) secreted in the milk of transgenic pigs was studied. Transgenes having different regulatory elements of the murine milk protein, whey acidic protein, were used with cDNA and genomic human protein C (hPC) DNA sequences to obtain lower and higher expressing animals. The cDNA pigs had a range of expression of about 0.1–0.5 g/l milk. Two different genomic hPC pig lines have expressed 0.3 and 1–2 g/l, respectively. The rhPC was first purified at yields greater than 60 per cent using a monoclonal antibody (mAb) to the activation site on the heavy chain of hPC. Subsequent immunopurification with a calcium‐dependent mAb directed to the γ‐carboxyglutamic acid domain of the light chain of hPC was used to fractionate a population having a higher specific anticoagulant activity in vitro. The higher percentages of Ca2+‐dependent conformers isolated from the total rhPC by immunopurification correlated well with higher specific activity and lower expression. A rate limitation in γ‐carboxylation of rhPC was clearly identified for the higher expressing animals. Thus, transgenic animals with high expression levels of complex recombinant proteins produced a lower percentage of biologically active protein.

Recombinant human protein C expression in the milk of transgenic pigs and the effect on endogenous milk immunoglobulin and transferrin levels.

Colostrum and milk are natural vehicles for acquiring passive immunity and are valuable tools for decreasing neonatant mortality from diarrheal disease. The effects of recombinant human protein C (rhPC) expression levels on endogenous immunoglobulin and transferrin content of the milk of different lineages of transgenic pigs were studied. The levels of rhPC in the milk ranged from 40 to 1200 μg/ml. Transgenic pigs with rhPC expression levels less than 500 μg/ml had no significant differences in milk protein composition with respect to nontransgenic pigs. A line of transgenic pigs having rhPC expression levels of 960–1200 μg/ml had two- to three-fold higher IgG, IgM, and secretory IgA concentrations compared to other transgenic and nontransgenic pig groups (P < 0.05), and four- to five-fold higher transferrin levels than nontransgenic pigs (P < 0.05). Changes in milk protein composition were not associated with mastitis or other pathologic disruption of epithelial cell junctions as indicated by normal casein and albumin levels in milk. Since IgG, IgM, secretory IgA, and transferrin are transported into the milk by transcytosis, higher levels of these proteins indicate that transcyctosis in the mammary epithelial cell was likely upregulated in pigs having high rhPC expression levels. This study is the first that shows a statistically significant example that mammary tissue specific expression of a heterologous protein can enhance endogenous phenotypic characteristics of milk.