Characterization of a variant of carcinoembryonic antigen (CEA) using monoclonal and polyclonal anti-CEA antibodies

A variant of the carcinoembryonic antigen (CEA) with lower molecular weight than a CEA reference preparation has been separated from CEA. Using a polyclonal, spleen absorbed anti-CEA antiserum, the variant crossreacts with reference CEA in immunodiffusion. The CEA-activity of the variant has been demonstrated using an enzyme-immunoassay with monoclonal CEA specific antibodies. There is sufficient immunological evidence that this variant is a distinct antigen different from the crossreactive antigens described so far. The reactivity of the polyclonal anti-CEA antiserum with the CEA variant was abolished by absorption against the immobilized variant.     

Identification and characterization of a human transthyretin variant

An apparent Mr variant of plasma transthyretin (TTR), previously detected using 2-D PAGE, is the first reported occurrence of this type of human TTR variant. We characterized the variant TTR to determine the nature of this difference. Comparative tryptic peptide maps of variant and normal TTR and sequencing of peptides which differed indicated the variant contained a single amino acid substitution of valine for tyrosine at position 116. Because such a change requires two nucleotide substitutions, we postulate the variant arose through mutation in codon 116 of a heretofore unrecognized polymorphic or rare variant allele of TTR.     

A variant of S49 mouse lymphoma cells with enhanced secretion of cyclic AMP.

A novel variant of S49 mouse lymphoma cells is described which is resistant to growth arrest and cytolysis by dibutyryl cyclic AMP but, in contrast to previously described variants, has normal cyclic AMP-dependent protein kinase. The variant is also resistant to N6-monobutyryl cAMP but is sensitive to killing by 8-bromo cAMP and cholera toxin. Extracts of the variant appear to contain wild type levels of both O2'-butyrylesterase and cyclic AMP phosphodiesterase activities. Accumulation of exogenous [3H]dibutyryl cyclic AMP is reduced in the variant suggesting a defect in either uptake or secretion of the analog or its metabolic products. Accumulation of cyclic AMP in variant cells after stimulation of adenylate cyclase with either isoproterenol or cholera toxin is also reduced compared with wild type cells, although cyclase activity of membranes prepared from the variant cells is normal. Extracellular accumulation of cyclic AMP after stimulation of variant cells with isoproterenol is greater than that found with wild type cells. It is concluded that the variant has an alteration in its cyclic AMP secretion mechanism resulting in more efficient extrusion of cyclic AMP than in wild type cells.     

Cyclic AMP-dependent phosphorylation of erythrocyte variant pyruvate kinase

Cyclic AMP-dependent phosphorylation of a variant erythrocyte pyruvate kinase (PK; EC 2.7.1.40) was studied. This variant PK shows a faster electrophoretic mobility than the normal enzyme. The decreased enzyme activity observed in this variant is associated with a quantitative decrease of enzyme protein. Other parameters are within normal ranges. The partially purified variant PK is phosphorylated with a subsequent increase of k0.5s (phosphoenolpyruvate) similar to the normal control, suggesting that the structural abnormality of the variant enzyme has no influence on the phosphorylation-deactivation mechanism. On the other hand, the variant PK in the erythrocyte was less extensively phosphorylated than PK in normal erythrocytes. This may be the result of abnormal metabolism in the patient's red cells, including increased 2,3-diphosphoglycerate and decreased adenosine triphosphate levels.     

The dermal-epidermal junction of human skin contains a novel laminin variant

We report the identification of a novel laminin variant that appears to be unique to a subset of epithelial basement membranes. The variant contains two chains electrophoretically and immunologically identical to the B1 and B2 chains. Epitopes contained in the laminin A chain are absent from the molecule, and a 190-kD chain substitutes for the A chain. V8 protease analysis and Western blotting studies indicate that the variant 190-kD chain shows structural and immunological similarity to the 200-kD chain of kalinin. Rotary shadowing analysis indicates that the 190-kD chain contributes a large globular structure to the variant long arm, but lacks the short arm contributed to laminin by the A chain. The variant is produced by cultured skin explants, human keratinocytes and a squamous cell carcinoma line, and is present in human amniotic fluid. Polyclonal antibodies raised to kalinin, a recently characterized novel component of anchoring filaments, and mAb BM165 which recognizes a subunit of kalinin (Rousselle et al., 1991) cross react with the variant under nonreducing conditions. Immunohistological surveys of human tissues using the crossreacting antikalinin antiserum indicate that the distribution of this laminin variant is at least restricted to anchoring filament containing basement membranes. We propose the name K-laminin for this variant.     

Reliably Detecting Clinically Important Variants Requires Both Combined Variant Calls and Optimized Filtering Strategies

A diversity of tools is available for identification of variants from genome sequence data. Given the current complexity of incorporating external software into a genome analysis infrastructure, a tendency exists to rely on the results from a single tool alone. The quality of the output variant calls is highly variable however, depending on factors such as sequence library quality as well as the choice of short-read aligner, variant caller, and variant caller filtering strategy. Here we present a two-part study first using the high quality ‘genome in a bottle’ reference set to demonstrate the significant impact the choice of aligner, variant caller, and variant caller filtering strategy has on overall variant call quality and further how certain variant callers outperform others with increased sample contamination, an important consideration when analyzing sequenced cancer samples. This analysis confirms previous work showing that combining variant calls of multiple tools results in the best quality resultant variant set, for either specificity or sensitivity, depending on whether the intersection or union, of all variant calls is used respectively. Second, we analyze a melanoma cell line derived from a control lymphocyte sample to determine whether software choices affect the detection of clinically important melanoma risk-factor variants finding that only one of the three such variants is unanimously detected under all conditions. Finally, we describe a cogent strategy for implementing a clinical variant detection pipeline; a strategy that requires careful software selection, variant caller filtering optimizing, and combined variant calls in order to effectively minimize false negative variants. While implementing such features represents an increase in complexity and computation the results offer indisputable improvements in data quality.     

Temperature-sensitive transport of glycoproteins to the surface of a variant mouse lymphoma cell line.

The expression of mouse mammary tumor virus (MMTV) glycoproteins on the surface of stably infected mouse lymphoma cell line W7MG1 is dramatically increased by glucocorticoid hormones. A variant cell line, W7M.TS1, was selected from W7MG1 for its lack of expression of MMTV glycoproteins on the cell surface in response to treatment with glucocorticoid. Hormonal stimulation of MMTV RNA levels and hormone-induced cytolysis occurred normally in the variant cells. Furthermore, the rates of production of the precursor and mature forms of MMTV glycoproteins in the presence of glucocorticoid were similar in variant and wild-type cells. However, the accumulation of MMTV glycoproteins on the cell surface after hormone treatment was delayed by about 8 h in the variant relative to wild-type cells. The steady-state level of a constitutively expressed cellular protein, T200, on the variant cell surface was comparable to that on wild-type cells. However, in pulse-chase experiments, the appearance of newly synthesized T200 on the cell surface was delayed in the variant compared with wild-type cells. Another glucocorticoid hormone response, removal of H-2 class I antigens from the cell surface, was also delayed in the variant relative to wild-type cells, suggesting that turnover or internalization of cell surface glycoproteins may also be affected in the variant. The defects in the variant cell line were observed at 37 degrees C, but not at 31 degrees C; the variant cells grew normally at both temperatures. This variant phenotype defines a new genetic entity that is important for transport of glycoproteins between internal microsomal compartments and the cell surface.     

ACP1GUA-1--a low-activity variant of human erythrocyte acid phosphatase: association with increased glutathione reductase activity.

ACP1GUA-1, a variant of human erythrocyte acid phosphatase, exists as a polymorphism (allele frequency of .132) in the Guaymi Indians of Central America. This variant has an electrophoretic mobility similar to the common B- and C-type variants, but individuals of the ACP1GUA-1 phenotype have a level of enzyme activity only 27% of the activity expected for the ACP1C variant. The GUA-1 variant is more thermostable than is the B variant, and the order of responsiveness to the modulation of activity by purine analogs and folate is always (B)-(A)-(GUA-1). Thus, the GUA-1 variant is a low-activity variant with C-like regulatory properties. Erythrocytes from individuals of the ACP1GUA-1 phenotype have increased basal levels of glutathione reductase, and a larger fraction of the glutathione reductase protein is present as the holoenzyme, indicating increased levels of flavin adenine dinucleotide in the erythrocytes of these individuals. This is consistent with the suggestion that ACP1 has a physiological function as a flavin mononucleotide phosphatase.     

A genetically determined variant of the a-subunit of lactic dehydrogenase in the deer mouse

Analysis by starch gel electrophoresis of erythrocyte hemolysates and brain extracts indicated that an electrophoretic variant of lactic dehydrogenase discovered in Peromyscus involves the A subunit. The tetramers containing the variant molecules migrate more rapidly than the normal ones, and the staining reactions suggested a reduced enzymatic activity. Comparisons of the erythrocyte LDH activity of normal and homozygous variant animals indicated that the variant enzyme is intrinsically less active than the normal. The variant is inherited as an autosomal codominant and appears to confer no selective disadvantage.This work was supported in part by a grant GM-15885 from the National Institutes of Health, U.S. Public Health Service, and in part by a supporting fund established in the name of Zachary Pitts Research Fellowship.     

A new variant of the anion transport protein in human erythrocytes

The major plasma membrane protein of human erythrocytes is the anion transport protein, termed protein 3. We previously reported a variant form of protein 3 that is elongated on the amino-terminal end of the molecule, which is exposed on the cytoplasmic side of the membrane, but otherwise its features are identical with those of the normal molecule. We have termed this molecule protein 3 variant 1. We now report a new variant form, protein 3 variant 2. The erythrocyte donor was a double heterozygote whose red cells possess a normal protein 3 and a protein 3 variant which is elongated and possesses a second variation at the 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) reactive site. Variant 2 reacts with 4,4'-diisothiocyano-1,2-diphenylethane-2,2'-disulfonic acid (H2DIDS) more readily than does the normal molecule. At high pH values, H2DIDS acts as a bifunctional cross-linking agent; it cross-links the proteolytic products generated by Pronase (or chymotrypsin) treatment of variant 2 less efficiently than noted for normal protein 3 or the first variant. Thus, the newly identified molecule has an alteration at the DIDS reactive site, which is near the outer surface of the membrane. The results can be interpreted as indicating that the DIDS binding site of variant 2 is more exposed than the normal molecule, but further removed from the site on the carboxyl-terminal fragment involved in cross-linking. Although there is a difference in the reactivity of the two protein 3 chains in variant 2, the reaction of variants 1 and 2 and normal cells with varying concentrations of [3H]H2DIDS results in the same amount of incorporation in all cells. Since protein 3 exists as a dimer or higher aggregate in the membrane, these results may indicate an interaction between monomers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tetramer formation of a variant type human transthyretin (prealbumin) produced by Escherichia coli expression system

A variant of human transthyretin(TTR, prealbumin) with methionine for valine substitution at position 30 is a major component of amyloid fibrils found in patients of familial amyloidotic polyneuropathy(FAP) type I, an autosomal dominant genetic disease. But the molecular nature of the variant TTR has been obscure, because most of plasma TTR from FAP patients is a mixture of variant and wild type TTR and no pure preparation of the variant has been available. For this reason, we constructed a system in which the variant type TTR was efficiently synthesized. In this system, the recombinant variant TTR was first synthesized as a fusion protein with E. coli outer membrane protein A (ompA) signal peptide, processed to eliminate the signal peptide and finally secreted to the culture medium. The final concentration of the recombinant variant TTR in the medium was about 5 mg/l. SDS polyacrylamide gel electrophoresis and gel filtration analysis suggested that the recombinant variant TTR can form tetramer as seen for native one. Purification of the protein was accomplished by only two steps of chromatography.     

Glucocorticoid-dependent maturation of mouse mammary tumor virus glycoproteins in mouse lymphoma cells: isolation of variants with constitutive viral protein maturation and normal glucocorticoid receptor function

The posttranslational maturation and cell surface localization of mouse mammary tumor virus (MMTV) envelope glycoproteins is regulated by glucocorticoid hormone in mouse T-lymphoma cell line W7MG1. Only when the cells are cultured with glucocorticoid is the MMTV envelope precursor, Pr74, converted efficiently to the two mature proteolytic products, gp52 and gp33. By immunological selection we have isolated protein-processing variants that express the mature viral proteins constitutively on the cell surface. The rate of synthesis of Pr74 is indistinguishable in variant and wild-type cells, but the variants efficiently convert Pr74 to gp52 and gp33 even when grown without the hormone. The variant phenotype persists when the variant cells are fused with uninfected wild-type cells to form somatic cell hybrids, indicating that the variant phenotype resulted from expression of a new or altered function that is not expressed in wild-type cells grown without glucocorticoid. Although the specific gene whose structure or regulation is altered in the variant has not yet been determined, some possibilities have been eliminated. First, the number and function of the glucocorticoid receptors in the variant cells was normal, suggesting that alterations in this protein were not responsible for the variant phenotype. Second, comparison by two-dimensional gel electrophoresis of gp52 produced in variant and wild-type cells revealed no differences in size or charge, indicating no gross differences in the processing of the viral proteins in the variant and wild-type cells.     

Report and characterization of a new variant, EB, of human red cell acid phosphatase.

A new variant phenotype of human red cell acid phosphatase, designated EB, was discovered in a male during a survey of blood donors from Copenhagen, Denmark. Electrophoretically, the variant revealed the two isozymes corresponding to the B type as well as two fast moving anodic isozymes. The enzyme activity and thermostability were found to be higher than in any earlier reported type. Isoelectric focusing of the variant type indicated that the isoelectric point of the variant enzyme is lower than in the common types.     

Identification of an alternative splicing variant of cathepsin C/dipeptidyl-peptidase I

Cathepsin C/dipeptidyl-peptidase I is a papain-like lysosomal cysteine proteinase implicated in the processing of various proenzymes to their active forms. In this study, we identified an alternative splicing variant of cathepsin C in both human and mouse species for the first time. The variant messenger RNA (mRNA) encodes 137 amino acids corresponding to the first and second exons, followed by additional 31 amino acids. The two newly recognized exons are located in the former intron 2. The variant mRNA is distributed ubiquitously, but predominantly in kidney, placenta, and lymph nodes. Furthermore, both interleukin 4 (IL-4) and IL-13, but not a range of cytokines induce expression of the variant in bronchial epithelial cells. These results indicate that the variant may play a role in regulating the biological activities of cathepsin C, involved in the pathogenesis of bronchial asthma.     

Defining the locus of origin of a genetically determined electrophoretic variant of a multilocus enzyme system; the calcutta-1 of human LDH system is a B-locus variant

Summary Six (four Hindus, one Sikh, and one Muslim) out of 213 individuals originating from different parts of the Indian subcontinent (namely, Andhra Pradesh, Maharashtra, Uttar Pradesh, East Punjab, and West Punjab) were found to be Calcutta-1 (CAL1) variants of lactate dehydrogenase (LDH). The CAL1 variant was originally described (and thus, generally believed at present) as an allelic variant at the LDHA locus in chromosome 11. By using an improved Cellogel electrophoretic procedure the isozyme patterns observed in the erythrocytes and leukocytes of the variant have indicated that the CAL1 is not a variant of LDHA but that of LDHB, a chromosome 12 marker. This suggestion was supported by the isozyme patterns of LDH in a set of segregating clones of man-mouse somatic cell hybrids with the variant as human partner. Moreover, the variant cosegregated consistently with the human chromosome 12 and with the markers firmly assigned to the latter but not with human chromosome 11 or its markers in these hybrids. These results confirmed that the CAL1 is an LDHB variant.     

Identification and characterization of a novel human APH-1b splice variant lacking exon 4

APH-1 is one of the four essential components of the presenilin-gamma-secretase complex and has two human homologs, APH-1a, and APH-1b, both of which are seven-pass membrane proteins. Here, we identified a novel splice variant of human APH-1b. This variant lacks exon 4, which encodes the entire fourth transmembrane domain. The mRNA expression of this variant was detected in most tissues at low levels. In transiently transfected cells, protein expression of the APH-1b variant was much lower than that of the wild-type. Furthermore, exogenous expression of the APH-1-interacting protein, nicastrin, significantly increased the variant protein levels. These data suggest that the APH-1b variant protein is destabilized, and implies that the fourth transmembrane domain plays an important role in the protein stability and function of APH-1.     

Mapping of segmental antigenic determinants on structurally related variant surface glycoproteins of Trypanosoma brucei

To study common and variant specific antigenic determinants on variant surface glycoproteins from Trypanosoma brucei, we have selected four serologically cross-reacting variant populations. Monoclonal antibodies were raised against the purified variant surface glycoproteins from each variant trypanosome population. Six monoclonal antibodies bind to segmental epitopes and one binds to a topographically assembled epitope. Amino acid compositions of these variant surface glycoproteins reveal striking conservation of certain residues including cysteine and charged amino acids. We also find that all seven monoclonal antibodies used in this study bind to protein determinants not exposed on the surface of the living trypanosome. Only one monoclonal antibody exhibits homologous specificity, while the remainder display cross-reactivity for three or all four variant surface glycoproteins. In addition, polyacrylamide gel electrophoresis peptide mapping and Western blots probed with each monoclonal antibody reveal significant peptide homologies. Furthermore, two pairs of monoclonal antibodies recognize two epitopes that are possibly immunodominant. The significance of these findings is discussed in terms of the structural similarities and differences among variant surface glycoproteins.     

Expression and modulation of CD44 variant isoforms in humans

CD44 is a ubiquitous surface molecule that exists as a number of isoforms, generated by alternative splicing of 10 "variant" exons. Little is known about the expression and function of the variant isoforms, except that certain isoforms may play a role in cancer metastasis. We produced mAbs against CD44 variant regions encoded by exons 4v, 6v, and 9v, by immunizing mice with a fusion protein spanning variant exons 3v to 10v. A comprehensive analysis of human tissues revealed that CD44 variant isoforms were expressed widely throughout the body, principally by epithelial cells. However there was differential expression of CD44 variant exons by different epithelia. Most epithelia expressed exon 9v, but much fewer expressed 6v or 4v. The regions of epithelia that expressed the highest levels of the variant isoforms were the generative cells, particularly the basal cells of stratified squamous epithelium, and of glandular epithelium. CD44 variant isoforms were also expressed differentially by leukocytes, with CD44-9v expressed at very low levels and CD44-6v and 4v virtually absent. However, CD44-9v and CD44-6v were the main variants that were transiently upregulated on T cells after mitogenic stimulation and on myelomonocytic cell lines by TNF alpha and IFN gamma treatment. Some epithelial cell lines could preferentially upregulate CD44-6v upon IFN gamma incubation. These results show that CD44 variant isoforms are expressed much more widely than first appreciated, and that expression of the variant isoforms on some cell types can be modulated by particular cytokines.     

Effect of Heat on Spores of Rough and Smooth Variants of Bacillus stearothermophilus

Spores of variants of Bacillus stearothermophilus were subjected to activating and lethal temperatures. Spore suspensions which were incubated longer contained a higher percentage of spores of the rough variant. The effect of sublethal heat on spore suspensions containing mixed variants (rough and smooth) was difficult to measure at sublethal temperatures (110 C), since the rough variant was not as heat-resistant. While the rough variant was activated in a shorter time, the smooth variant was not activated; when the smooth variant was activated, the rough was killed. A higher percentage of the smooth variant was forced into dormancy after being held at 50 C for 30 hr than the rough variant. When mixed populations were subjected to a lethal temperature (120 C), the curves only reflected the smooth variant. Since the curves which represented the smooth variant or mixtures containing the smooth variant were not linear, this was thought to be due to activation overbalancing the lethal effect. This research emphasized the importance of variants in explaining differences in spore resistance among spore suspensions of the same strain.