Alex Zhu

Anti-N-glycolylneuraminic acid antibodies identified in healthy human serum

Abstract: The first and major clinical obstacle in xenotransplantation is antibody‐mediated hyperacute rejection. Although human natural antibodies against Galα1,3Gal (Gal) antigens, which are common on porcine cells and organs, have been identified to play a major role in hyperacute rejection, other natural antibodies against non‐Gal epitopes may be also involved in the process. Here, we present evidence suggesting that the majority of human anti‐non‐Gal antibodies are specific for carbohydrate structures carrying terminally linked N‐glycolylneuraminic acid (NeuGc), a xenoantigen existing in almost all animals except humans. Furthermore, this anti‐NeuGc activity is detectable in 85% of healthy humans, implicating the involvement of NeuGc in hyperacute rejection and the importance of developing strategies for removing NeuGc for clinical xenotransplantation.

Cloning and functional expression of a cDNA encoding coffee bean α-galactosidase

Abstract Purified coffee bean α-galactosidase (αGal) has been used for removing terminal α-galactose residues from the glycoconjugates at the red cell surface, in studies of blood group conversion. Here, we report the isolation and sequence of the full-length clone for coffee bean αGal by using the polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) techniques. The cDNA clone (1.4 kb) contains a single open reading frame which encodes a protein of 378 amino acids (aa). Its authenticity is confirmed by perfect alignment of aa sequences obtained from purified coffee bean αGal, and by immune reaction with the antibody raised against the enzyme. Furthermore, the protein produced in insect cells shows enzymatic activity towards a synthetic αGal substrate, p -nitro-phenyl- α -galactopyranoside.

The synthesis, testing and use of 5-fluoro-α-D-galactosyl fluoride to trap an intermediate on green coffee bean α-galactosidase and identify the catalytic nucleophile.

Abstract 5-Fluoro-α- d -galactopyranosyl fluoride was synthesized and its interaction with the active site of an α-galactosidase from green coffee bean ( Coffea arabica ), a retaining glycosidase, characterized kinetically and structurally. The compound behaves as an apparently tight binding ( K i =600 nM) competitive inhibitor, achieving this high affinity through reaction as a slow substrate that accumulates a high steady-state concentration of the glycosyl-enzyme intermediate, as evidenced by ESiMS. Proteolysis of the trapped enzyme coupled with HPLC/MS analysis allowed the localization of a labeled peptide that was subsequently sequenced. Comparison of this sequence information to that of other members of the same glycosidase family revealed the active site nucleophile to be Asp145 within the sequence LKY Ḏ NCNNN. The importance of this residue to catalysis has been confirmed by mutagenesis studies.

Use of RhD fusion protein expressed on K562 cell surface in the study of molecular basis for D antigenic epitopes

The human D antigens, one of the most clinically important blood groups, are presented by RhD protein with a putative 12 transmembrane topology. To understand the molecular basis for the complex antigenic profile of RhD protein, we expressed a series of RhD fusion proteins using different portions of Duffy protein as a tag in erythroleukemic K562 cells. Because the reactivity of monoclonal anti-RhD antibody, LOR15C9, depends mainly on the sequence coded by exon 7 of RhD, we altered DNA sequence corresponding to the amino acid residues 323–331(A) and 350–354(B) in the exon 7. The mutation in region B resulted in a severe reduction in LOR15C9 binding by flow cytometry analysis, suggesting that region B may play an important role in constituting antigen epitopes recognized by LOR15C9. On the other hand, a slight decrease in the antibody binding was observed for the region A mutant, suggesting that the intracellularly located region A may elicit a long distance effect on the formation of exofacial antigen epitopes. In addition, using various monoclonal antibodies against RhD, we compared the antigenic profile of expressed RhD fusion protein with that of endogenous RhD in K562 cells as well as in erythrocytes.

Identification of tyrosine 108 in coffee bean α-galactosidase as an essential residue for the enzyme activity

The cDNA for coffee bean alpha-galactosidase (alpha-Gal) has been cloned and expressed in a baculovirus expression system. An early study of coconut alpha-Gal by chemical modification suggested that one tyrosine residue is at or near the active site. In order to identify such a critical residue, we replaced two tyrosine residues (positions 108 and 158) with phenylalanine by site-directed mutagenesis. The mutated DNA strands, as well as the wild-type ones, were subcloned into pVL vector and transformed into Sf9 insect cells for intracellular expression. The replacement of Tyr-158 with phenylalanine resulted in a mutant alpha-Gal (Y158F) which retained approx. 88% of the activity of wild-type enzyme. However, the substitution of Tyr-108 by phenylalanine (Y108F) almost abolished the enzymatic activity (1.8% of wild-type activity). The Vmax/Km value for the mutant Y108F was 0.027, which was over a 1000-fold lower than that of wild-type alpha-Gal. Our data suggest that Tyr-108 is critical for the enzymatic activity of alpha-Gal.

Trp-16 is essential for the activity of α-galactosidase and α-N-acetylgalactosaminidase

Abstract By expressing site-directed mutants in the methylotrophic yeast strain Pichia pastoris, the role of a tryptophan residue at position 16 in the activity of α-galactosidase and α- N -acetylgalactosaminidase, two closely related exoglycosidases, was studied. A substitution of Trp-16 with an arginine residue in α- N -acetylgalactosaminidase abolished the enzyme activity, which was confirmed by replacing a 600 bp fragment containing the mutation with the corresponding wild-type sequence. The same tryptophen residue was then substituted with an alanine in both enzymes by site-directed mutagenesis to reveal a possible relationship between their active sites. The purified α- N -acetylgalactosaminidase mutant demonstrated a specific activity of 2.8 × 10 −2 U/mg and a V max / K m of 4.3 × 10 −2 , which were both more than a thousandfold lower than corresponding values for the wild-type enzyme. Furthermore, the mutant failed to bind to an affinity resin, suggesting the involvement of Trp-16 in substrate-binding. In addition, the purified α-galactosidase mutant resulted in more than a 10 4 -fold decrease in specific activity. Thus our data suggest that Trp-16 in both α-galactosidase and α- N -acetylgalactosaminidase is critical for enzymatic activity, which in turn supports the hypothesis that these two enzymes may share a catalytic mechanism involving similar residues in their active sites.

Initial investigation of the potential of modified porcine erythrocytes for transfusion in primates

Abstract:  There is a shortage of human blood for transfusion. The possibility of using α‐galactosidase‐treated pig red blood cells (pRBCs) for transfusion into humans has been investigated. pRBCs were treated in vitro with α‐galactosidase. In vitro binding of antibodies (Abs) in baboon or human sera to untreated/treated pRBCs was assessed by flow cytometry and serum cytotoxicity. In vivo clearance rates of (1) autologous baboon red blood cells (RBCs), (2) unmodified pRBCs, and (3) α‐galactosidase‐treated pRBCs were measured after transfusion into baboons receiving either no treatment or depletion of complement ± depletion of anti‐Galα1–3Gal (Gal) Ab or of macrophage phagocytes. In vitro binding of baboon or human Abs to treated pRBCs was absent or minimal compared with untreated pRBCs, and serum cytotoxicity was completely inhibited. In vivo autologous baboon RBCs survived for >16 days and unmodified pRBCs for <15 min in an untreated baboon. Treated pRBCs survived for 2 h in an untreated baboon, for 24 h in a complement‐depleted baboon, and for 72 h when the baboon was depleted of both complement and anti‐Gal Ab, or of complement and macrophage phagocytes. All baboons, however, became sensitized to Gal antigens. Failure to prolong the in vivo survival of treated pRBCs could be due to inadequate removal of Gal epitopes because sensitization to Gal developed, or could imply other, as yet unidentified, causes for RBC destruction. To fully assess the potential of pRBC transfusion in humans, more complete α‐galactosidase treatment of pRBCs will be required.

Introduction to porcine red blood cells: Implications for xenotransfusion

Advances in the field of xenotransplantation raise the intriguing possibility of using porcine red blood cells (pRBCs) as an alternative source for blood transfusion. The domestic pig is considered the most likely donor species for xenotransplantation. However, identification of xenoantigens on porcine erythrocytes and elucidation of their possible roles in antibody-mediated RBC destruction are necessary for developing clinical strategies to circumvent immunological incompatibility between humans and pigs. Although the alphaGal epitope (Galalpha1,3Galbeta1,4GIcNAc-R) is the major xenoantigen on porcine erythrocytes and is responsible for the binding of the majority of human natural antibodies, other non-alphaGal xenoantigens have been identified. The importance of these non-alphaGal xenoantigens in binding human natural antibodies and subsequently triggering immunological responses cannot be underestimated. Our data suggest that non-alphaGal xenoantigen(s) identified on the porcine erythrocyte membrane are not only recognized by xenoreactive human natural antibodies but are also involved in complement-mediated hemolysis.

Cloning, functional expression and purification of endo-β-galactosidase from Flavobacterium keratolyticus

Endo-beta-galactosidase (EC 3.2.1.103) is an enzyme that hydrolyzes internal endo-beta-galactosyl linkages in keratan sulfate, and glycoconjugates with N-acetyl-lactosamine repeating units. Here, we report the cloning of the endo-beta-galactosidase-encoding gene from Flavobacterium keratolyticus, its expression in Escherichia coli and the purification of the enzyme. The enzyme was purified over 15000-fold to apparent homogeneity. The purified endo-beta-galactosidase consists of a single band of about 43kDa on SDS-PAGE and has a specific activity of 148micro/mg. Based on peptide sequences derived from the purified enzyme, a full-length clone encoding endo-beta-galactosidase was isolated from F. keratolyticus genomic DNA. The gene contains a single open reading frame coding for a protein of 422 amino acid residues with a putative N-terminal signal peptide. Its authenticity was confirmed by colinearity of deduced amino acid sequences with the peptide sequences, and synthesis of enzyme in E. coli.