Functional display of proteins, mutant proteins, fragments of proteins and peptides on the surface of filamentous (bacterio) phages: A review.

Cytoplasmic expression of complex eukaryotic proteins inEscherichia coli usually yields inactive protein preparations. In some cases, (part) of the biological activity can be recovered by rather inefficient denaturation-renaturation procedures. Recently, novel concepts have been developed for the expression of fully functional eukaryotic proteins inE. coli. Essential to the success of these procedures is the transport of such proteins across the inner membrane to the periplasmic space, allowing proper folding and the establishment of disulfide bonding. Subsequently, fully functional proteins can be exposed on the surface of filamentous (bacterio)phages, provided a system is employed that consists of a cloning vector (e.g. the phagemid pComb3, Barbas et al., 1991) that generates phage particles in the presence of a helper phage. The main advantage of surface display of recombinant proteins is to facilitate the screening of very large numbers of different molecules by simple selection methods ("panning"). In addition, periplasmic expression yields relatively large quantities (e.g. 1 mg l(-1) of culture) soluble protein. In this review, the principle aspects of this novel expression system based on the phagemid pComb3 will be discussed. Two examples for functional periplasmic expression of human proteins inE. coli will be presented, namely i) the antigen-binding moiety (Fab fragment) of human immunoglobulins (IgGs) and ii) the human plasminogen activator inhibitor 1, an essential regulator of the plasminogen activation system. Finally, perspectives for the application of this system to express mutant proteins, fragments of proteins and peptides are indicated.

Characterization of N-cadherin messenger RNA and polypeptide expression in rat.

The cell adhesion molecule N-cadherin is a member of the cadherin gene superfamily. The protein is involved in morphogenetic processes, including neurite extension. In this study, N-cadherin mRNA and polypeptide expression were investigated in rat brain, liver, muscle, heart, kidney and lung during postnatal development and aging. Six synthetic oligonucleotide probes covering different parts of mouse N-cadherin cDNA all hybridized to 5.2, 4.3-4.4 and 3.5 kb mRNAs in rat tissues. The mRNA pattern differed between tissues and, furthermore, the amount of N-cadherin mRNA and polypeptides in brain, liver and heart was higher than in muscle, kidney and lung. N-cadherin expression decreased slightly during early postnatal development in all tissues, whereas no changes in N-cadherin expression were observed during aging. Antibodies against a fusion protein containing the transmembrane and cytoplasmic sequence of chick N-cadherin were produced. These antibodies, termed anti-N-cad-cyt, were compared to the R-156 antibodies which recognize the 24 C-terminal amino acids of N-cadherin and which have been shown to react with a broad spectrum of cadherins. Using these two antibodies, it was shown that the 130 kDa N-cadherin polypeptide was subject to calcium-dependent cleavage of the cytoplasmic domain. Conversely, in the absence of calcium the polypeptide was cleaved extracellularly, producing two C-terminal fragments of 85 and 95 kDa. A 122 kDa polypeptide was recognized by both antibodies and may be either an alternatively spliced form of N-cadherin or a closely related cadherin.

Age-related changes in expression of neural cell adhesion molecule (NCAM) in heart: a comparative study of newborn, adult and aged rats.

The neural cell adhesion molecule (NCAM) has been implicated in cellular interactions involved in cardiac morphogenesis and innervation. In this study, expression of NCAM mRNA and protein was characterized in rat heart during postnatal development and aging (postnatal days 1, 10, 40, 270, and 730). Alternative splicing of NCAM mRNA was analyzed by Northern blotting using DNA oligonucleotide probes designed for demonstration of certain exons or exon combinations. Total NCAM mRNA was downregulated during postnatal development followed by upregulation in the aging heart. Three major NCAM mRNA classes of 6.7, 5.2 and 2.9 kb were expressed in newborn heart in approximately equal proportions. At all other ages, the mRNAs of 5.2 and 2.9 kb were more predominant than the 6.7 kb mRNA. During postnatal development and aging, expression of exon VASE was selectively downregulated in the 6.7 kb NCAM mRNA class, whereas it was clearly detectable in NCAM mRNA classes of 5.2 and 2.9 kb. Insertion of exons a and AAG between exons 12 and 13 was more pronounced in the 5.2 and 2.9 kb NCAM mRNAs than in the 6.7 kb mRNA at all ages. Insertions at the 12/13 junctions decreased in the 6.7 kb mRNA as compared to the 5.2 and 2.9 kb mRNAs during postnatal development. Quantification of NCAM protein by enzyme-linked immunosorbent assay showed that NCAM protein amount decreased from a level of 0.93 microgram NCAM/mg total protein at birth to postnatal day 40 where a level of 0.21 microgram NCAM/mg total protein was found. During aging, NCAM increased again to 0.34 micrograms NCAM/mg total protein at postnatal day 730. NCAM isoforms were studied by immunoblotting analysis. At all ages, the major NCAM isoforms of 190, 145 and 120 kDa were observed in heart, the 145 kDa isoform being predominant.(ABSTRACT TRUNCATED AT 250 WORDS)

Different isoforms and stock-specific variants of the cell adhesion molecule C-CAM (cell-CAM 105) in rat liver.

C-CAM is a cell adhesion molecule of the immunoglobulin superfamily with homophilic binding properties. Here we used the polymerase chain reaction to isolate clones of C-CAM from a rat liver cDNA library. Sequence analyses identified two major isoforms, C-CAM1 and C-CAM2, which differed in their 3' ends. C-CAM2 lacked a sequence of 53 nucleotides that was present in C-CAM1. This causes a frame shift and new stop codons, which gives rise to cytoplasmic domains of different sizes in the two isoforms (10 versus 71 amino-acid residues). In addition, all the clones had a different nucleotide and deduced amino-acid sequence (variant b) in the most N-terminal of the four immunoglobulin-like domains, compared to a previously published C-CAM sequence (variant a). Northern-blot analyses with specific oligonucleotide probes demonstrated that two different rat stocks expressed either variant a or variant b. Northern-blot analyses of rat liver and lung also showed that at least five different C-CAM transcripts are produced. Two major mRNA size classes of 4.0 kb and 6.0 kb, and one minor class of 3.0 kb were found. Both the 4.0-kb and 3.0-kb messenger classes reacted with two different probes that could distinguish between C-CAM1 and C-CAM2, while the 6.0-kb population only reacted with the probe selective for C-CAM1. Taken together these data demonstrate the existence of four different protein-coding sequences of rat liver C-CAM (C-CAM1 a and b, and C-CAM2 a and b). We suggest that both allelic variation and alternative splicing may contribute to the isoform-expression pattern of C-CAM in rats.

Age-related changes in expression of the neural cell adhesion molecule in skeletal muscle: a comparative study of newborn, adult and aged rats.

Neural cell adhesion molecule (NCAM) is expressed by muscle and involved in muscle-neuron and muscle-muscle cell interactions. The expression in muscle is regulated during myogenesis and by the state of innervation. In aged muscle, both neurogenic and myogenic degenerative processes occur. We here report quantitative and qualitative changes in NCAM protein and mRNA forms during aging in normal rat skeletal muscle. Determination of the amount of NCAM by e.l.i.s.a. showed that the level decreased from perinatal to adult age, followed by a considerable increase in 24-month-old rat muscle. Thus NCAM concentration in aged muscle was sixfold higher than in young adult muscle. In contrast with previous reports, NCAM polypeptides of 200, 145, 125 and 120 kDa were observed by immunoblotting throughout postnatal development and aging, the relative proportions of the individual NCAM polypeptides remaining virtually unchanged at all ages examined. However, changes in the extent of sialylation of NCAM were demonstrated. Even though the relative amounts of the various NCAM polypeptides were unchanged during aging, distinct changes in NCAM mRNA classes were observed. Three NCAM mRNA classes of 6.7, 5.2 and 2.9 kb were present in perinatal and young adult skeletal muscle, whereas only the 5.2 and 2.9 kb mRNA classes could be demonstrated in aged muscle. This indicates that metabolism of the various NCAM polypeptides is individually regulated during aging. Alternative splicing of NCAM mRNA in skeletal muscle was studied by Northern blotting using DNA oligonucleotide probes specifically hybridizing to selected exons or exon combinations. Exon VASE, which has previously been shown to be present in both brain and heart NCAM mRNA, was virtually absent from skeletal muscle at all ages studied. In contrast, the majority of NCAM mRNA in postnatal skeletal muscle was shown to contain extra exons inserted between exons 12 and 13. Of the various possible exon combinations at this splice site, the combinations 12-a-AAG-13 and 12-a-b seemed to be prevalent in postnatal skeletal muscle. No significant change in the relative proportion of these two exon combinations occurred during aging. The observed upregulation of NCAM protein in aged muscle supports the assumption that an increasing proportion of muscle fibres are denervated in aged muscle. Selective upregulation of the 5.2 and 2.9 kb mRNA forms have previously been demonstrated in muscle cell lines and in primary cultures of muscle cells during formation of myotubes in vitro, and this switch in NCAM mRNA classes has been suggested to correlate with myogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of NCAM mRNA and polypeptides in aging rat brain.

In aging brain, degenerative as well as compensatory regenerative processes are believed to occur. The neural cell adhesion molecule NCAM is involved in developmental and regenerative processes in the brain. However, the role of NCAM in aging brain has not been characterized. In this study, the expression of NCAM mRNAs and polypeptides was investigated in aging rat brain. The 7.4 and 6.7 kb NCAM mRNAs were selectively downregulated during postnatal development, and the 5.2 and 2.9 kb NCAM mRNAs were upregulated. However, from postnatal day 40 to old age no change in NCAM mRNA classes was observed. The fraction of NCAM mRNA containing the VASE exon increased postnatally but remained stable during adult life. VASE, which is believed to modulate the binding capacity, seemed to be relatively more abundant in the 7.4 and 6.7 kb NCAM mRNAs, encoding transmembrane NCAM forms, than in the 5.2 and 2.9 kb NCAM mRNAs, coding for glycosyl phosphatidylinositol (GPI) linked NCAM. Conversely, insertion of exons a and AAG between exons 12 and 13, a region containing two fibronectin type III repeats, seemed to be more pronounced in 5.2 and 2.9 kb NCAM mRNAs than in the 7.4 and 6.7 kb mRNAs. During postnatal development an increase in the fraction of 6.7 kb NCAM mRNA containing the exons a and AAG was observed. However, during aging the fraction of NCAM mRNAs containing this exon combination seemed constant. At the protein level, NCAM-A was downregulated both during development and aging. No changes were observed during aging in the composition of soluble NCAM forms in the brain, cerebrospinal fluid or blood plasma. The amount of NCAM in rat brain decreased during postnatal development, but remained at a constant level from postnatal day 40 to old age. To conclude, several changes in NCAM expression occur during early postnatal development emphasizing the important role of this molecule in the morphogenetic processes. During aging, a significant selective downregulation of NCAM-A was observed indicating that in general only minor regenerative processes occur in the brain.

Molecular biology of cadherins in the nervous system.

Cadherins are cell-cell adhesion molecules belonging to the Ca(2+)-dependent cadherin superfamily. In the last few years the number of cadherins identified in the nervous system has increased considerably. Cadherins are integral membrane glycoproteins. They are structurally closely related and interspecies homologies are high. The function is mediated through a homophilic binding mechanism, and intracellular proteins, directly or indirectly connected to the cadherins and the cytoskeleton, are necessary for cadherin activity. Cadherins have been implicated in segregation and aggregation of tissues at early developmental stages and in growth and guidance of axons during nervous system development. These functions are modified by changes in type(s) and amount of cadherins expressed at different developmental stages. The regulatory elements guiding cadherin expression are currently being elucidated.

Demonstration of L1-related mRNAs in rat brain using DNA oligonucleotide probes.

Only one copy of the cell adhesion molecule L1 gene is present in the mouse genome, and only one mRNA of 6 kilobases (kb) is expressed in mouse brain [1987, Neurosci. Lett. 82, 89-94]. We have constructed 5 synthetic oligonucleotide probes covering different parts of the published mouse L1 cDNA sequence. Using these probes 3 distinct mRNAs of 9.0, 7.0 and 6.0 kb in rat brain could be demonstrated. Hybridizations performed at different stringency conditions indicated that the 9.0 and 7.0 kb mRNAs were highly related to the L1 mRNA of 6.0 kb expressed in rat brain. The 7.0 kb mRNA is possibly coding for a rat homologue of chicken Nr-CAM, whereas the 9.0 kb mRNA may code for a new member of the L1 family.

Characterization of NCAM expression and function in BT4C and BT4Cn glioma cells.

The neural cell adhesion molecule, NCAM, plays an important role in cell-cell adhesion. Therefore, we have studied NCAM expression in the glioma cell lines BT4C and BT4Cn. We demonstrate that the 2 cell lines differ in their metastatic ability; while BT4C cells have a very low capacity for producing experimental metastases, that of BT4Cn cells is high. In BT4C cells NCAM is synthesized as 4 polypeptides with Mr's of 190,000, 140,000, 115,000 and 97,000. The 140,000, 115,000 and 97,000 polypeptides are glycosylated and for the 140,000 and 115,000 polypeptides sulfatation is observed. Conversely, no NCAM protein synthesis is observed in BT4Cn cells, even though NCAM mRNA is expressed. Thus, development of an increased metastatic capacity is accompanied by the disappearance of NCAM protein expression in this model system. The functional importance of NCAM expression was studied by a cell-substratum binding assay in which the binding of BT4C and BT4Cn cells to NCAM immobilized to glass was assessed. We found that BT4C cells adhere specifically to NCAM, and that adhesion is inhibited by anti-NCAM Fab'-fragments, while no specific binding of BT4Cn cells to NCAM was observed. The BT4C and BT4Cn cell lines thus constitute an important new model system for the study of tumor invasion and metastasis and of the role of cell adhesion molecules in these processes.

Characterization of rat brain NCAM mRNA using DNA oligonucleotide probes.

A number of different isoforms of the neural cell adhesion molecule (NCAM) have been identified. The difference between these is due to alternative splicing of a single NCAM gene. In rat brain NCAM mRNAs with sizes of 7.4, 6.7, 5.2, 4.3 and 2.9 kb have been reported. We have synthesized six DNA oligonucleotides, that hybridize to different exons in the NCAM gene. Furthermore we have constructed three oligonucleotides, that exclusively hybridize to mRNAs lacking certain exons, by letting them consist of sequences adjacent to both sides of the splice sites. By means of these probes we have characterized the five NCAM mRNAs in rat brain.

Demonstration of immunochemical identity between the synaptic vesicle proteins synaptin and synaptophysin/p38.

The synaptic vesicle proteins synaptin and synaptophysin/p38 were shown to be immunochemically identical. Western immunoblot analysis of Triton X-100 extracts from rat brain showed that polyclonal polyspecific anti-synaptin antibodies and monoclonal antibody SY38 against synaptophysin both reacted with a band of 38 kDa. In two-dimensional immunoblots of chromaffin granule membranes from bovine adrenal medulla anti-synaptin and anti-synaptophysin antibodies also recognized the same component. Finally, in a Western immunoblotting experiment SY38 reacted with an immuno-isolated synaptin antigen.