Anna Krop-Watorek

A molecular approach for isolating high‐affinity Fab fragments that are useful in blood group serology

BACKGROUND: Multiple mouse hybridoma antibodies recognize the antigens of the MNS blood group system. The Fab fragments of several of these antibodies were expressed on bacteriophage and as soluble proteins. The parental N92 anti‐N IgG monoclonal antibody (parental N92 MoAb), but not its monovalent, soluble Fab fragment (N92 Fab fragment), agglutinated antigen‐positive red cells by an antiglobulin method. Light‐chain shuffling was used to isolate mutant N92 Fab fragments with higher affinity that would function by agglutination.

The dimeric structure of carcinoembryonic antigen (CEA)

The results of SDS-polyacrylamide gel electrophoresis and cross-linking experiments indicated that carcinoembryonic antigen is a dimer composed of two identical or closely similar noncovalently bound subunits, dissociating on heating in the presence of SDS. The dissociation is reversible upon the detergent removal.

The subunit structure of non-specific cross-reacting antigen (NCA)☆

NCA was purified from normal human lung (L-NCA) and from liver metastases of colon adenocarcinoma (T-NCA), L-NCA and T-NCA had different carbohydrate compositions, but showed an immunological identity in double immunodiffusion with the use of anti-CEA and anti-L-NCA sera. In SDS-polyacrylamide gel electrophoresis L-NCA and T-NCA showed similar molecular weights of about 110,000 and 120,000, respectively. However, if the samples were heated for 3 min at 100 degrees C in the presence of 1% SDS before electrophoresis, their apparent mol. wt decreased to about 50,000, suggesting the dissociation of NCA molecules into subunits. The dissociation of NCA was independent of the presence of mercaptoethanol and did not destroy the ability of NCA to precipitate with anti-CEA and anti-NCA sera. Besides NCA (and CEA in tumor tissue), the presence of a lower molecular weight cross-reacting antigen was observed in lung and tumour. Double immunodiffusion showed that this additional antigen was not a dissociated form of NCA.

Adhesion of human uroepithelial cells to E-selectin : Possible involvement of sialosyl LewisA-ganglioside

In a previous study we showed that tumorigenic and invasive human uroepithelial cell lines are characterized by the presence of sialosyl Lea (sLea) ganglioside. Our data suggested that expression of this glycolipid correlated with acquisition of the malignant phenotype by human urothelial cells. To evaluate the postulated adhesion function of sLea antigen, we studied the adherence of 6 human urothelial cell lines with different expressions of this carbohydrate structure to E‐selectin‐expressing CHO cells. The only cell line that bound specifically to E‐selectin was Hu 1703He, which expressed the highest level of sLea antigen. The involvement of carbohydrate‐E‐selectin interaction in the adhesion of Hu 1703He cells was indicated by the following facts: (i) anti‐E‐selectin monoclonal antibody (MAb) completely abolished binding to E‐selectin‐expressing CHO cells; (ii) removal of sialic acid from Hu 1703He cells highly decreased the adhesion. Adhesion correlated with the presence of several sLea‐carrying glycoproteins, which was shown by immunoblotting of Hu 1703He cell lysate with anti‐sLea MAb 19‐9. The binding of antibody was abolished when cell lysate was treated with O‐sialoglycoprotein endopeptidase, suggesting that sLea is present on O‐linked oligosaccharides. However, incubation of Hu 1703He cells with O‐sialoglycoprotease had no effect on adhesion to E‐selectin or on binding of 19‐9 MAb to the cell surface. Our data suggest that (i) protein‐bound sLea oligosaccharides represent only a minor portion of whole sLea antigen produced by uroepithelial cells; (ii) effective binding to E‐selectin occurs when sLea oligosaccharide present on cell‐surface glycosphingolipids is expressed in high density since the cell lines with moderate expression of sLea ganglioside did not bind to E‐selectin‐transfected CHO cells.

Isolation of carcinoembryonic antigen N-terminal domains (N-A1) from soluble aggregates.

Carcinoembryonic antigen (CEA) was identified as a prominent tumor-associated antigen in human colorectal cancer and it is still intensively investigated. However, its physiological role remains unclear. The CEA molecule is composed of seven highly hydrophobic, immunoglobulin-like domains, six of which contain a single disulphide bridge. The production of recombinant protein containing Ig-like domains in bacterial expression systems often results in partial degradation or insolubility due to aggregation hampering the analysis of their native structure and function. Here, we present a new method of expression and purification of CEA N-terminal domains (N-A1) fused to MBP in Escherichia coli. In order to optimize the expression and purification of CEA N-A1 domains we evaluated bacteria cultivation conditions, the length of N-A1 domains, fusion systems (GST- and MBP-tag), IPTG concentrations and protein purification conditions. We have found that MBP-N-A1 fusion protein digested with TEV protease forms soluble aggregates composed of N-A1 domains and incompletely digested MBP-N-A1 fusion protein. Using 1.25 M guanidinium chloride (GdmCl) as a component of the elution buffer we were able to achieve an almost complete dissociation of the aggregates. The dissociation was monitored by circular dichroism and fluorescence measurements. The CD spectra and Ellmans assay suggest that the conformation of N-A1 domains and their disulphide bonds are correct.

Construction of an agglutination tool: recombinant Fab fragments biotinylated in vitro.

The pComb3H vector system is used for constructing and panning recombinant antibody libraries. It allows for expression of monovalent Fab fragments, either on the surface of M13 phage, or in the form of soluble proteins secreted into the periplasmic space of bacteria. We constructed a modified pComb3H vector containing cDNA encoding for a 23-amino acid fragment of the Escherichia coli biotin carboxy carrier protein (BCCP), which is an acceptor sequence for biotinylation. The vector was used to express the Fab fragment recognizing human glycophorin A. The purified Fab fragment containing this biotin acceptor sequence was effectively biotinylated in vitro using biotin ligase (BirA). The specificity and avidity of the biotinylated Fab fragments were similar to the previously produced, unmodified Fab fragments. An avidin-alkaline phosphatase conjugate was used to detect the recombinant Fab fragments, instead of secondary antibody. In addition, when biotinylated Fab fragments were mixed with avidin, red blood cells were directly agglutinated.

Behavior in immunoblotting of dimeric and monomeric forms of nonspecific cross-reacting antigen (NCA-50) from normal lung

The dimeric and monomeric forms of NCA-50 from normal lung showed differences in binding to nitrocellulose in an immunoblotting procedure. The NCA dimer showed weaker binding, it passed through nitrocellulose and was more easily washed out from the blot. The NCA monomer was bound strongly and was detected with higher sensitivity following immunoblotting.

Single nucleotide polymorphisms in A4GALT spur extra products of the human Gb3/CD77 synthase and underlie the P1PK blood group system

Contrary to the mainstream blood group systems, P1PK continues to puzzle and generate controversies over its molecular background. The P1PK system comprises three glycosphingolipid antigens: Pk, P1 and NOR, all synthesised by a glycosyltransferase called Gb3/CD77 synthase. The Pk antigen is present in most individuals, whereas P1 frequency is lesser and varies regionally, thus underlying two common phenotypes: P1, if the P1 antigen is present, and P2, when P1 is absent. Null and NOR phenotypes are extremely rare. To date, several single nucleotide polymorphisms (SNPs) have been proposed to predict the P1/P2 status, but it has not been clear how important they are in general and in relation to each other, nor has it been clear how synthesis of NOR affects the P1 phenotype. Here, we quantitatively analysed the phenotypes and A4GALT transcription in relation to the previously proposed SNPs in a sample of 109 individuals, and addressed potential P1 antigen level confounders, most notably the red cell membrane cholesterol content. While all the SNPs were associated with the P1/P2 blood type and rs5751348 was the most reliable, we found large differences in P1 level within groups defined by their genotype and substantial intercohort overlaps, which shows that the P1PK blood group system still eludes full understanding.