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S L Spitalnik

Publications and source records attributed to S L Spitalnik.

At least 19 recordsLinked to original sources
PubMed2002

Section 5: Structural/genetic analysis of mAbs to blood group antigens. Coordinator's report.

The heavy and light chain immunoglobulin variable region nucleotide sequences for 219 mAbs to human red blood cells were collected from workshop participants, published reports, and Genbank. Information regarding antigen specificity, species of origin, method of cloning, and other relevant serological properties was correlated with the sequence data. Immunoglobulin sequences were analyzed to determine the heavy- and light-chain immunoglobulin genes used and the overall extent of somatic mutation from germline configuration. Approximately 50% of the sequences encoded antibodies with Rh(D) specificity with the remaining sequences encoding mAbs to other Rh-related antigens, antigens of the ABO, MNS, and Kell blood group systems, and several others. Surprisingly, no sequence data were available for mAbs with specificity for a number of common Rh antigens, common Kell antigens, or antigens of the Lewis, Kidd, or Duffy blood group systems. The majority of mAbs were of human origin but included a significant number of macaque mAbs, murine mAbs, and a small number of synthetically-designed recombinant antibodies. Both cellular (EBV-transformation, cell fusion) and molecular (phage display) approaches were used for antibody cloning. Analysis of certain groups of sequences demonstrated patterns of immunoglobulin gene restriction, repertoire shift, and somatic mutation. Analysis of other mAbs demonstrated the value of antibody sequence data for the design and production of novel reagents useful in blood group serology.

Animals↗
PubMed2001

Characterization of galactosyl glycerolipids in the HT29 human colon carcinoma cell line.

Glycoglycerolipids constitute a family of glycolipids with apparently very restricted expression in human tissues. They have previously been detected only in the testis and the nervous system. In the present study, two glycoglycerolipids were isolated from the HT29 human colon carcinoma cell line. The glycoglycerolipids were structurally characterized as a monogalactosylglycerolipid (1-O-alkyl-2-O-acyl-3-O-(beta-galactosyl)-sn-glycerol) and a digalactosylglycerolipid (1-O-alkyl-2-O-acyl-3-O-(beta-galactosyl(1-4)alpha-galactosyl)-sn-glycerol) using NMR and mass spectrometry. This digalactosylglycerolipid has not previously been structurally characterized. When HT29 cells were allowed to differentiate into more enterocyte-like cells by culture in glucose-free medium, expression of both of these glycoglycerolipids was greatly diminished. The presence of glycoglycerolipids in a human colon carcinoma cell line indicates that expression of this family of glycolipids may not be as restricted as previously thought. Instead this class of glycolipids may serve as differentiation antigens in various normal tissues and in tumor development. The Galalpha1-4Gal epitope was previously identified as a receptor for bacterial adhesins and toxins. The finding that this epitope is also linked to a glycerolipid moiety opens up new possible roles for this carbohydrate receptor in intracellular signaling.

Carbohydrate Metabolism↗
PubMed1999

Study of the sugar chains of recombinant human amyloid precursor protein produced by Chinese hamster ovary cells.

The N- and O-glycans of recombinant amyloid precursor protein (APP), purified from Chinese hamster ovary cells transfected with the human 695-amino acid form of APP, were separately released by hydrazinolysis under different conditions. The reducing ends of the released N- and O-glycans were reduced with NaB3H4 and derivatized with 2-aminobenzamide (2AB), respectively. After acidic N-glycans were obtained by anion-exchange column chromatography, these were converted to neutral oligosaccharides by sialidase digestion, demonstrating that their acidic nature was entirely due to sialylation. The sialidase-treated N-glycans were then fractionated by lectin column chromatography and their structures were determined by linkage-specific sequential exoglycosidase digestion. These results demonstrated that recombinant APP has bi- and triantennary complex type N-glycans with fucosylated and nonfucosylated trimannosyl cores. In a similar fashion, the 2AB-labeled O-glycans derived from APP were determined to be mono- and disialylated core type 1 structures. Taken together, these results indicate that recombinant APP has sialylated bi- and triantennary N-glycans with fucosylated and nonfucosylated cores and sialylated O-glycans with core type 1 structures.

Amyloid beta-Protein Precursor↗
PubMed1999

A novel, non-nested reverse-transcriptase polymerase chain reaction (RT-PCR) test for the detection of the t(15;17) translocation: a comparative study of RT-PCR cytogenetics, and fluorescence In situ hybridization.

BACKGROUND: The development of a rapid and simple reverse-transcription polymerase chain reaction (RT-PCR) assay is described that identifies the promyelocytic leukemia- retinoic acid receptor alpha (PML-RARa) hybrid messenger RNA (mRNA), a characteristic feature of acute promyelocytic leukemia (APL). METHODS AND RESULTS: Randomly primed complementary (cDNA) is synthesized from leukocyte RNA and amplified in the presence of Taq Gold in 2 separate reaction tubes containing primer pairs specific for intron 3 (bcr 3, long [L] form mRNA transcript) and intron 6 (bcr 1, short [S] form)/exon 6 (bcr 2, variant [V] form) breakpoints in PML, respectively. The different sized products generated from each RNA transcript (S, L, or V forms) are readily and unambiguously distinguishable after agarose gel electrophoresis without the need for either nested PCR or hybridization. The sensitivity of the assay is 1 in 10,000 to 1 in 100,000. The separate amplification of a b2-microglobulin transcript controls for adequate RNA and cDNA preparation. The newly developed assay was used clinically for the evaluation of 78 patients with APL. It was rapid and more sensitive than cytogenetic karyotyping, both for the diagnosis of APL and the assessment of minimal residual disease (MRD) after therapy. RT-PCR detected PML-RARa mRNA in all cases positive for the t(15;17) translocation by cytogenetics. However, as many as 50% and 80% of the diagnostic specimens and the specimens for MRD assessment, respectively, that were positive by RT-PCR were negative by cytogenetics. The ratio of cases with L-form to S-form PML-RARa fusion transcript was 2:1, whereas 3 cases (10%) had fusion sites in exon 6 of the PML gene (V forms). In addition, approximately 50% of the patients were diagnosed morphologically with microgranular M3V-type leukemia, but no significant correlation with PML breakpoints was found. CONCLUSION: The current assay is rapid, sensitive, and specific without using nested PCR or hybridization.

Biomarkers, Tumor↗
PubMed1999

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. STUDY DESIGN AND METHODS: Light-chain cDNA libraries, constructed from mice immunized with N-type glycophorin A, were inserted into a recombinant pComb3H vector containing the N92 Fd fragment. The N92 Fd fragment:light-chain libraries were panned on N-type glycophorin A or NN red cells, and antigen-binding clones were isolated. Purified parental N92 MoAb and the Fab fragments were evaluated by enzyme-linked immunosorbent assay and agglutination. RESULTS: The novel NNA7, C1, and G11 Fab fragments all bound to N-type glycophorin A with higher affinity than did the N92 Fab fragment. The affinity of the library-derived clones was equivalent to that of the parental N92 MoAb. Although their fine specificity differed slightly from the parental N92 MoAb, the clones functioned equivalently by agglutination using an antiglobulin method. CONCLUSIONS: Light-chain shuffling allowed the isolation of bacterially produced, high-affinity, soluble, monovalent recombinant anti-N Fab fragments that functioned well by agglutination. This approach is useful in obtaining inexpensive serologic reagents that may replace conventional MoAbs produced by tissue culture methods.

Agglutination↗
PubMed1998

The amino acid following an asn-X-Ser/Thr sequon is an important determinant of N-linked core glycosylation efficiency.

Many eukaryotic proteins are modified by Asn-linked (N-linked) glycosylation. The number and position of oligosaccharides added to a protein by the enzyme oligosaccharyltransferase can influence its expression and function. N-Linked glycosylation usually occurs at Asn residues in Asn-X-Ser/Thr sequons where X not equal Pro. However, many Asn-X-Ser/Thr sequons are not glycosylated or are glycosylated inefficiently. Inefficient glycosylation at one or more Asn-X-Ser/Thr sequons in a protein results in the production of heterogeneous glycoprotein products. These glycoforms may differ from one another in their level of expression, stability, antigenicity, or function. The signals which control the efficiency of N-linked glycosylation at individual Asn residues have not been fully defined. In this report, we use a site-directed mutagenesis approach to investigate the influence of the amino acid at the position following a sequon (the Y position, Asn-X-Ser/Thr-Y). Variants of rabies virus glycoprotein containing a single Asn-X-Ser/Thr sequon at Asn37 were generated. Variants were designed with each of the twenty common amino acids at the Y position, with either Ser or Thr at the hydroxy (Ser/Thr) position. The core glycosylation efficiency of each variant was quantified using a cell-free translation/glycosylation system. These studies reveal that the amino acid at the Y position is an important determinant of core glycosylation efficiency.

Amino Acid Sequence↗
PubMed1998

Only selected light chains combine with a given heavy chain to confer specificity for a model glycopeptide antigen.

The M and N human blood group glycopeptide Ags are carried on RBCs by glycophorin A. Previous results suggested that the murine humoral immune response against the N, but not the M, Ag is restricted. In addition, these results suggested that particular highly homologous heavy chains might be able to combine promiscuously with various light chains to yield anti-N specificity. To examine this, the current study used Fab phage methodology to couple an array of light chains, obtained from cDNA libraries isolated from immunized mice, to single Fd obtained from N61, N92, and 425/2B hybridomas. Interestingly, for the chimeric Fab to retain M or N specificity, the new light chains needed to belong to the same Vk gene family as the light chain from the parental, hybridoma-derived mAb. In some cases the new light chains modified the Fab affinity and fine specificity. For example, library-derived light chains coupled with the N92 Fd yielded chimeric Fab with increased affinity. In particular, the affinity of these univalent chimeric Fab for the N Ag was equivalent to that of the bivalent parental IgG mAb. Taken together, these results demonstrate that particular structures formed by the light chain V region are required to cooperate with a particular heavy chain V region to create a functional binding site for these glycopeptide Ags. They also demonstrate a lack of heavy chain promiscuity in the formation of murine anti-M and anti-N Abs.

Amino Acid Sequence↗
PubMed1998

The role of site-specific N-glycosylation in secretion of soluble forms of rabies virus glycoprotein.

Rabies virus glycoprotein is important in the biology and pathogenesis of neurotropic rabies virus infection. This transmembrane glycoprotein is the only viral protein on the surface of virus particles, is the viral attachment protein that facilitates virus uptake by the infected cell, and is the target of the host humoral immune response to infection. The extracellular domain of this glycoprotein has N-glycosylation sequons at Asn37, Asn247, and Asn319. Appropriate glycosylation of these sequons is important in the expression of the glycoprotein. Soluble forms of rabies virus glycoprotein were constructed by insertion of a stop codon just external to the transmembrane domain. Using site-directed mutagenesis and expression in transfected eukaryotic cells, it was possible to compare the effects of site-specific glycosylation on the cell-surface expression and secretion of transmembrane and soluble forms, respectively, of the same glycoprotein. These studies yielded the surprising finding that although any of the three sequons permitted cell surface expression of full-length rabies virus glycoprotein, only the N-glycan at Asn319 permitted secretion of soluble rabies virus glycoprotein. Despite its biological and medical importance, it has not yet been possible to determine the crystal structure of the full-length transmembrane form of rabies virus glycoprotein which contains heterogeneous oligosaccharides. The current studies demonstrate that a soluble form of rabies virus glycoprotein containing only one sequon at Asn319 is efficiently secreted in the presence of the N-glycan processing inhibitor 1-deoxymannojirimycin. Thus, it is possible to purify a conformationally relevant form of rabies virus glycoprotein that contains only one N-glycan with a substantial reduction in its microheterogeneity. This form of the glycoprotein may be particularly useful for future studies aimed at elucidating the three-dimensional structure of this important glycoprotein.

Animals↗
PubMed1997

Recombinant blood group antigens are useful for studying the fine specificity of antibodies against glycophorin A encoded antigens.

Recombinant wild-type and variant forms of glycophorin A (GPA) were expressed in wild-type and glycosylation-defective Chinese hamster ovary cells. The binding to recombinant GPA of antibodies submitted to the Third International Workshop was assessed by immunoprecipitation and indirect immunofluorescence. This is a powerful approach for determining the fine specificity of antibodies to blood group antigens. The advantages and limitations of this approach are discussed.

Animals↗
PubMed1996

The role of glycosylation in synthesis and secretion of beta-amyloid precursor protein by Chinese hamster ovary cells.

Alzheimer's disease is characterized by beta-amyloid deposition in the brain. This peptide is derived by proteolytic cleavage from beta-amyloid precursor protein (APP), a highly glycosylated membrane glycoprotein containing both N- and O-glycans. There are three major isoforms of APP, which are derived by alternative splicing and contain 695, 751, or 770 amino acids. Since glycosylation can affect many properties of glycoproteins, we studied the role of N- and O-glycosylation in the synthesis and secretion of APP. APP expression was examined in untransfected wild-type, Lec-8 mutant, and ldlD mutant Chinese hamster ovary (CHO) cells and in analogous clonal cell lines expressing either the transfected human wild-type 695-amino-acid form of APP (APP695-WT) or a form mutated to delete N-glycosylation sites (APP695-XX). These studies showed that maturation of APP in CHO cells is accompanied by the addition of multiple short O-glycans with the following structures: Neu5Acalpha2-3Galbeta1-3GalNAc, Neu5Acalpha2-3Galbeta1-3[Neu5Acalpha2-6]GalNAc, and GalNAc. Using glycosylation-defective mutant CHO cell lines and soluble inhibitors of glycosylation, we found that APP secretion was diminished when core N-glycosylation or N-glycan processing was blocked. Surprisingly, similar results were found when synthesis and secretion of either APP695-WT or APP695-XX were analyzed. These results indicate that defective N-glycosylation of other cellular proteins, but not of APP itself, affects the metabolism of APP. Interestingly, inhibition of O-glycosylation did not affect the biosynthesis or secretion of APP. The results of these studies may shed some light on the role that protein glycosylation may play in the pathogenesis of Alzheimer's disease.

Amyloid beta-Protein Precursor↗
PubMed1996

The amino acid at the X position of an Asn-X-Ser sequon is an important determinant of N-linked core-glycosylation efficiency.

N-Linked glycosylation is a common form of protein processing that can profoundly affect protein expression, structure, and function. N-Linked glycosylation generally occurs at the sequon Asn-X-Ser/Thr, where X is any amino acid except Pro. To assess the impact of the X amino acid on core glycosylation, rabies virus glycoprotein variants were generated by site-directed mutagenesis with each of the 20 common amino acids substituted at the X position of an Asn-X-Ser sequon. The efficiency of core glycosylation at the sequon in each variant was quantified in a rabbit reticulocyte lysate cell-free translation system supplemented with canine pancreas microsomes. The presence of Pro at the X position completely blocked core glycosylation, whereas Trp, Asp, Chi, and Leu were associated with inefficient core glycosylation. The other variants were more efficiently glycosylated, and several were fully glycosylated. These findings demonstrate that the X amino acid is an important determinant of N-linked core-glycosylation efficiency.

Amino Acid Sequence↗
PubMed1996

Purification of a secreted form of recombinant rabies virus glycoprotein: comparison of two affinity tags.

Expression of recombinant eukaryotic proteins in transfected mammalian cell lines has become an important approach for the characterization of the structure and function of these proteins. However, it is often difficult to recover and purify the recombinant proteins. Therefore, the use of fusion proteins incorporating epitope or affinity tags has become more widespread. In this paper, we directly compare two affinity tags, the hexahistidyl tag and the biotin peptide mimetic, Strep-tag, for use in purification of a recombinant soluble form of rabies virus glycoprotein secreted by transfected Chinese hamster ovary fibroblasts. The recombinant rabies virus glycoproteins are denoted RGP(WT)T441his and RGP(WT)T443s-tag, respectively. These affinity tags were chosen because the chromatographic matrices (Ni(II)-NTA-agarose and recombinant core streptavidin-agarose, respectively) were readily available and these methods offered the possibility of a one-step purification using mild elution conditions. However, in our hands, neither method allowed for a one-step purification protocol. Nonetheless, it was possible to purify RGP(WT)T441his to homogeneity from crude conditioned medium using a combination of metal-chelate affinity chromatography and immunoaffinity chromatography. In contrast, although the Strep-tag has been useful for purifying recombinant proteins expressed in bacteria, we were not able to effectively purify RGP(WT)T443s-tag from conditioned medium using chromatography on recombinant core streptavidin-agarose.

Affinity Labels↗
PubMed1996

Blood type AB in the feline AB blood group system.

OBJECTIVE: To assess the genetics, frequency, and biochemistry of the AB blood type in cats. ANIMALS: Domestic shorthair and purebred cats in a breeding colony and privately owned catteries and blood samples from a large feline blood typing laboratory. PROCEDURES: Samples from cats with blood type AB were selected from the feline blood typing laboratory at the university. Breeding experiments and family studies were used for the genetic analysis of cats with blood type AB. Simple slide hemagglutination assays were used to type cats. Hemagglutination assays, flow cytometry, and ganglioside analysis by high-performance thin layer chromatography were applied to characterize the AB antigens. RESULTS: Type AB was rare (13/9,239 cats; 0.14% frequency) in cats of the United States and Canada. Type AB occurred only in breeds in which type B was also detected. Cats with type-AB blood express biochemical features of type-A and type-B antigens. Genetic analyses of families with blood type-AB cats are consistent with the hypothesis of 3 alleles: A, B, and AB. The AB allele is recessive to the A allele, but dominant over the B allele. There may be an additional genetic mechanism responsible for the inheritance of blood type AB in cats. CONCLUSION: Blood type AE is an extremely rare and separately inherited type in the feline AB blood group system. CLINICAL RELEVANCE: Kittens with type-AB blood born to queens with type-B blood are at similar risk for neonatal isoerythrolysis as kittens with type-A blood because anti-A alloantiserum from blood type-B queens recognizes AB red blood cells. Furthermore, cats with type-AB blood are best transfused with type-AB or type-A blood.

ABO Blood-Group System↗
PubMed1995

Immune responses to nucleic acid vaccines to rabies virus.

A plasmid vector expressing the full-length rabies virus glycoprotein (G protein) under the control of the simian virus 40 (SV40) promoter has previously been shown to induce upon intramuscular (i.m.) inoculation into mice a specific B- and T-cell-mediated immune response and protection against challenge with a virulent strain of the virus. Here we tested two parameters that might affect the efficacy of this DNA vaccine. First, we replaced the SV40 promoter of the original vector with the early promoter derived from cytomegalovirus leaving all other parameters of the plasmid intact. Although upon transfection in vitro the two vectors showed a striking difference in their ability to cause stable expression of the rabies virus G protein, upon i.m. inoculation into mice both constructs induced comparable immune responses. Second, we constructed a vector that induces expression of a secreted form of rabies G protein by inserting a stop codon just upstream of the transmembrane domain of the rabies G protein gene. The immune responses to the DNA vaccines expressing the two different forms of the G protein, secreted and membrane bound, were compared and found to be similar in magnitude. The long-term effect of DNA vaccination was also investigated especially with regard to adverse immunological reactions such as the induction of unresponsiveness against rabies virus and the development of antibodies to DNA. DNA vaccination was found to induce long-lasting immunity to rabies virus without apparent negative side effects such as development of T cell tolerance or generation of anti-DNA antibodies.

Animals↗
PubMed1995

Stable secretion of a soluble, oligomeric form of rabies virus glycoprotein: influence of N-glycan processing on secretion.

Rabies virus glycoprotein (RGP) is a 505 amino acid type I transmembrane glycoprotein that is important in the pathogenesis of rabies virus infection. RGP also stimulates the development of neutralizing antibodies by the host. N-linked glycosylation is required for both cell surface expression and immunogenicity of RGP. In the current study, a soluble form of RGP, constructed by insertion of a stop codon external to the transmembrane domain, was expressed in transfected Chinese hamster ovary cells. The soluble form of RGP was found to be appropriately antigenic and immunogenic. Similar to full-length RGP, the soluble form was assembled into homodimers and homotrimers. Core glycosylation was required for secretion of soluble RGP and cell surface expression of full-length RGP. In addition, initial glucose trimming of the N-glycans was necessary and sufficient for secretion of soluble RGP and cell surface expression of full-length RGP. Further N-glycan processing was not required for secretion or cell surface expression of soluble or full-length RGP, respectively.

Animals↗
PubMed1995

Construction of bacteriophage expressing mouse monoclonal Fab fragments directed against the human MN glycophorin blood group antigens.

BACKGROUND: The MN human blood group antigens are complex glycopeptide antigens at the amino terminus of glycophorin A. Many different mouse monoclonal antibodies to these antigens have been produced and characterized. The construction of combinatorial immunoglobulin libraries displaying antibody Fab fragments on the surface of bacteriophage (Fab-phage) represents a novel approach for developing monoclonal reagents, for exploring the diversity of the immune response to specific antigens, and for understanding the molecular basis of the interaction of an antibody with its antigen. However, it is necessary to determine whether Fab fragments displayed on bacteriophage surfaces retain immunologic characteristics similar to the intact antibodies. STUDY DESIGN AND METHODS: Fab-phage were constructed from three anti-N (AH7, N61, and N92) and two anti-M (425/2B and M2A1) murine hybridomas. The Fab-phage and parental hybridomas were compared by enzyme-linked immunosorbent assay, Western blotting, and flow cytometry. RESULTS: In each case, the Fab-phage and its parental hybridoma antibody had similar immunologic characteristics. In particular, their dependence on the pH of the buffer and on sialylation of the target antigen was similar. CONCLUSION: These results suggest that Fab-phage may provide novel reagents with applications in immunohematology and may be useful in the study of the immune response to human blood group antigens.

Animals↗