In Vitro Synthesis of Carbohydrate-Binding Proteins by Human Leucocytes

Allen, Herbert B.; Kachurek, Dave; Bulera, Steve; Kisailus, Edward C.; DiCioccio, Richard A.

doi:10.3109/08820138609094138

Cited by 6 publications

(8 citation statements)

References 24 publications

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“…With adequate reduction and alkylation of samples, this antiserum reacted with only a 14.5-kDa polypeptide present in splenic and HL-60 cell extracts and also reacted with galaptin purified from buffy coat and B lymphoblastoid cells. sponding to the previously reported 18.5-kDa galactosidebinding protein synthesized by HL-60 cells (Allen et al" 1986).…”

Section: Discussionsupporting

confidence: 82%

“…The studies reported here were carried out to obtain a better understanding of galaptin and its expression in human leucocytes and to determine if the metabolically labeled buffy coat cell lactose-binding CBP previously described (Allen, 1986;Allen et al, 1986) was related to the galaptin monomer. Despite the fact that buffy coat cells are a very heterogeneous population, they synthesize in vitro a major galactoside-binding monomeric CBP of M, 15.5-16.5K.…”

Section: Discussionmentioning

confidence: 99%

“…For in vitro lectin synthesis by buffy coat cells, crude buffy coat cells were purchased from the American Red Cross and further purified by dextran sedimentation to remove RBC. The washed buffy coat cells were incubated with [3H] leucine as previously described (Allen et al, 1986) or were incubated with [3H]leucine (10 /xCi/mL)/[35S]methionine (10 /tCi/mL) for 48 h in serum-free medium at 9 X 106 cells/mL.…”

Section: Methodsmentioning

confidence: 99%

“…Galaptin was adsorbed to DEAE-Sephacel for storage at -20 °C (Sharma et al, 1990). Purity and molecular weight were assessed by FPLC on Superose 12 and SDS-PAGE (Allen et al, 1987a), by Bio Gel P-30 chromatography (Allen et al, 1986), and by RP-HPLC and IEF (Sharma et al, 1990).…”

Section: Methodsmentioning

confidence: 99%

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Synthesis, isolation, and characterization of endogenous .beta.-galactoside-binding lectins in human leukocytes

Allen

Gottstine²,

Sharma³

et al. 1991

Biochemistry

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Galaptin, a beta-galactoside-binding lectin, was isolated from human buffy coat cells (peripheral leukocytes) and spleen by affinity chromatography. The molecular weight (32K) of the native buffy coat galaptin was similar to that for splenic galaptin. Their subunit molecular weight (14.5K), pI (4.60-4.85), and amino acid composition were identical. Both galaptins showed the presence of a single polypeptide when subjected to reversed-phase HPLC. Monospecific rabbit polyclonal antiserum raised against the 14.5-kDa subunit of splenic galaptin reacted with a 14.5-kDa polypeptide present in buffy coat cells, Epstein-Barr virus-immortalized B lymphoblastoid cells, and HL-60 promyelocytic leukemia cells. However, galaptin was not synthesized in vitro by buffy coat cells. Rather, a monomeric beta-galactoside-binding protein of Mr 15.5-16.5K that is immunologically distinct from galaptin was synthesized. This galactoside-binding protein was separable from galaptin by polyacrylamide gel electrophoresis and by anion-exchange chromatography. In contrast, immunoprecipitation experiments confirmed that galaptin was synthesized by the B lymphoblastoid cells. cDNA corresponding to the B lymphoblastoid cell mRNA encoding galaptin was amplified by the polymerase chain reaction. The amplified product was partially sequenced, and 299 nucleotides were identified. The derived amino acids corresponded to residues 6-65, 84-114, and 118-126 found to be present in human splenic galaptin. Immunohistochemical analyses revealed that galaptin was distributed throughout the cytoplasm of B lymphoblastoid cells rather than being localized to the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Discussionsupporting

confidence: 82%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Synthesis, isolation, and characterization of endogenous .beta.-galactoside-binding lectins in human leukocytes

Allen

Gottstine²,

Sharma³

et al. 1991

Biochemistry

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“…Lectin was isolated by affinity chromatography on lactosc-Sepharose 6B [31] or asialofctuin-Sepharose 2B [32]. Dialyzed extracts were loaded on affinity col umns.…”

Section: Methodsmentioning

confidence: 99%

Galactoside-Binding Lectin in Human Tissues

Allen

Piver

et al. 1987

Tumor Biol

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Lactose-inhibitable lectin activity has been analyzed by hemagglutination assay in a variety of human tissues and cells obtained at surgery and autopsy. The lectin activity was detected in surgically removed melanoma, sarcoma, colon carcinoma, breast carcinoma, adjacent non-malignant tissues, non-malignant tissues obtained at autopsy and in cells isolated from malignant effusions. Although, on average, malignant tissue had a higher hemag-glutinating titer than non-malignant tissue, similar tissues from different individuals varied widely in their apparent lectin content. The lectin was isolated from lung by affinity chro-matography and was found to have a native molecular mass of 31,000 daltons and a subunit molecular mass of 14,000 daltons. Utilizing rabbit anti-lung lectin serum in an immunohis-tochemical assay, the lectin was found to be distributed throughout the cytoplasm of lung epithelial cells. Ouchterlony immunodiffusion analysis confirmed the presence of this lectin in a variety of tissues and in some body fluids. In vitro metabolic radiolabelling experiments showed that the presence of lectin in tissues was most likely due to endogenous synthesis rather than absorption from body fluids. Lectin isolated from several tissues was found to bind to human buffy coat cell receptors.

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Lineage- and differentiation-dependent alterations in the expression of receptors for glycoconjugates (lectins) in different human hematopoietic cell lines and low grade lymphomas

Gabius

Hellmann

Ciesiolka

et al. 1989

Blut

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Important biological functions and cellular recognition phenomena are supposedly governed by specific sugar-protein interactions. Human hematopoietic cell lines offer an excellent model for the study of the expression of endogenous receptors for the carbohydrate part of glycoconjugates with respect to cell lineage and modulation by differentiation. Initially, a panel of fluorescent (neo)glycoproteins was successfully employed to demonstrate cytologically the actual presence of such receptors on different cell lines: the B lymphoblast line, Daudi; the T cell lymphoblastic leukemia line, P12; the multipotent leukemic line, K562 and the promyelocytic line, HL060. Biochemical analyses were performed using affinity chromatography on supports with immobilized lactose and asialofetuin (simple or complex beta-galactosides), melibiose (alpha-galactoside), fucose, N-acetyl-D-galactosamine, maltose (alpha-glucoside), the mannose-rich yeast glycoprotein, mannan, glycopeptides containing sialic acid residues and heparin. Subsequently, sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis was used to detect cell lineage-dependent changes in theses parameters. Differentiation-dependent changes in the expression of receptors with specificity to galactose, N-acetylgalactosamine, maltose and heparin were similarly uncovered upon dimethyl sulfoxide-induced differentiation of HL60 cells. Differences in this type of cellular characteristic were also apparent for lymphoma cells from patients with various histological subtypes of lowgrade lymphomas. This initial description of lineage- and differentiation-dependent differences in various human hematopoietic cell lines and in cells from patients with lowgrade lymphomas suggests that advances in the knowledge of the composition of endogenous sugar receptors (lectins) may aid in understanding aspects of the biological behavior of hematopoietic cells and their related malignancies via participation of sugar-protein (lectin) interactions.

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In Vitro Synthesis of Carbohydrate-Binding Proteins by Human Leucocytes

Cited by 6 publications

References 24 publications

Synthesis, isolation, and characterization of endogenous .beta.-galactoside-binding lectins in human leukocytes

Synthesis, isolation, and characterization of endogenous .beta.-galactoside-binding lectins in human leukocytes

Galactoside-Binding Lectin in Human Tissues

Lineage- and differentiation-dependent alterations in the expression of receptors for glycoconjugates (lectins) in different human hematopoietic cell lines and low grade lymphomas

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