Purification and partial characterization of the erythrocyte Kx protein deficient in McLeod patients.

A 37-kDa protein was immunopurified from human erythrocytes as a complex with a monoclonal antibody directed against the Kell blood group protein of 93 kDa. A rabbit antibody raised against the purified complex reacted on a Western blot with the 93-kDa and 37-kDa proteins and was able to immunoprecipitate the 37-kDa component from K0 erythrocytes which express large amount of the Kx antigen, but not from erythrocytes of patients suffering from McLeod syndrome, a X-linked disorder in which the Kx antigen is lacking. Additional studies have shown that the 37-kDa protein is not glycosylated, and permitted the sequence of the 22 first N-terminal amino acids to be established. This sequence was identical to the predicted protein product of the XK gene cloned recently, which is deleted or mutated in McLeod patients [Ho, M., Chelly, J., Carter, N., Danek, A., Crocker, P. & Monaco, A. P. (1994) Cell 77, 869-880]. Our findings provide strong evidence that the 37-kDa red cell membrane protein is identical to the Kx protein produced by the XK structural gene and demonstrate that Kx and Kell proteins are two subunits expressed as a complex hold by disulfide bond(s) at the red cell surface.

Influence of the size of the polar head of non-ionic detergents on membrane proteins immunoaffinity purification.

Nonionic polyoxyethylene type detergents (CxEy) are widely used to solubilize and purify membrane proteins. The detergent hydrophobic moiety (Cx) replaces phospholipids at exposed hydrophobic regions of the membrane proteins. During chromatography on an immobilized anti-Kell antibody to purify Kell protein (an integral erythrocyte protein), it was observed that the size of the polar head of an non ionic detergent added to the mobile phase appeared to influence the interaction of the detergent-protein complex with the immobilized antibody. Further studies were performed using another erythrocyte membrane protein, Glycophorin C and three anti-GPC monoclonal antibodies directed against three epitopes of the extracytoplasmic domain of the protein. The interaction of GPC with the three Protein A-coupled monoclonal antibodies was studied in the presence of three detergents C12E<9>, C13E<15> and C12E<23>. It was observed in batch mode and in column chromatography experiments that the adsorption of GPC to the immunoaffinity supports decreased as the size of the detergent polar head increased. Thus, the polyoxyethylene chain of a detergent might prevent the interaction of the detergent-protein complex with the immobilized antibody.

The LW blood group glycoprotein is homologous to intercellular adhesion molecules.

The LW blood group antigens reside on a 42-kDa erythrocyte membrane glycoprotein that was purified by immunoaffinity and partially sequenced. From this information, a specific PCR-amplified DNA fragment was used to screen a lambda gt11 human bone marrow cDNA library. Two forms of cDNA were isolated; the first encoded a single spanning transmembrane protein of 270 amino acids, including a 29-amino acid peptide signal and four potential N-glycosylation sites, and the second encoded a shortened protein form of 236 residues devoid of transmembrane and cytoplasm domains. A rabbit antibody raised against the 15 N-terminal amino acids of the predicted protein reacted on immunoblots with authentic LW glycoprotein and in indirect agglutination test with all human erythrocytes except those from LW(a-b-). This showed that the protein encoded by these clones was LW gene product and suggested that the N terminus of the LW protein is oriented extracellularly. Most interestingly, the LW protein was found to exhibit sequence similarities (with approximately 30% identity) with intercellular adhesion molecules ICAM-1, -2, and -3, which are the counter-receptors for the lymphocyte function-associated antigens LFA-1. The extracellular domain of LW consists, like that of ICAM-2, of two immunoglobulin-like domains, and the critical residues involved in the binding of LFA-1 to ICAMs were partially conserved in LW.

Affinity of trypsin for amidine derivatives immobilized on dextran-coated silica supports.

The pancreas contains two very analogous enzymes: trypsin and chymotrypsin. These two enzymes are very similar in their physicochemical characteristics and are therefore quite difficult to separate by classical purification procedures. They constitute a good model for affinity chromatography. It was previously demonstrated that amidine derivatives are able to interact strongly and specifically with these serine proteases and are often used as ligand in affinity chromatography. To understand the trypsin interaction mechanism, we synthesized different amidines and immobilised them with or without spacer arm on silica beads previously coated by dextran substituted with a calculated amount of positively charged diethylaminoethyl functions, in order to minimize the non-specific interactions of silanol groups of the silica material. First the affinity constant and the adsorption capacity of these supports for trypsin were determined in batch procedures, then they were used in affinity chromatography. The effects of ionic strength, pH and competitive inhibitors on proteins desorption were also studied. Last, to demonstrate the importance of passivation, the chromatographic performances of dextran-coated silica phases and a commercial support grafted with the same amidine were compared.

Specific adsorption of serine proteases on coated silica beads substituted with amidine derivatives.

Amidine derivatives interact with serine proteases, the inhibition being due to interactions between amidine functions and the active sites of the enzymes. Five different types of amidine (substituted or unsubstituted) were coupled to coated silica beads, which had previously been coated with DEAE-dextran to minimize the non-specific interactions due to silanol groups. Coated silica functionalized with substituted amidines shows a strong affinity towards human plasmin. This affinity is probably due to hydrophobic interactions between the substituted amidine and the human plasmin structure. Coated silica grafted by p-aminobenzamide gives a specific interaction with human plasmin. The importance of ionic strength and the steric conformation of the ligand is discussed. This support was used to purify thrombin from crude preparations by high-performance affinity chromatography.