Purification and partial characterization of a malignancy-associated glycoprotein

The isolation from cancer patient serum of a glycoprotein (Cc) associated with the presence of a variety of malignancies was previously reported. Although preliminary chemical and physical data indicated that Cc was different from identified circulating glycoproteins, subsequent immunological studies suggested that it was closely related to alpha 1-acid glycoprotein. Further analysis revealed the presence of two components in some Cc preparations and prompted a re-examination of the isolation and characterization data. In the present study, Cc was purified by a modified protocol which involved the use of pleural fluid obtained from individuals with cancer, and an alpha 1-acid glycoprotein antibody column to remove contaminating alpha 1-acid glycoprotein. Typically, the material not retained by the antibody column gave a single band with Mr 53,000 when subjected to sodium dodecyl sulfate-polyacrylamide electrophoresis. Amino terminal analysis revealed that the protein contained a blocked amino terminus, and carbohydrate analysis indicated that complex, asparagine-linked saccharide units were present. The product could be distinguished from alpha 1-acid glycoprotein and other previously described circulating glycoproteins by several criteria, including molecular weight, isoelectric point, and amino acid and carbohydrate composition. One of three preparations isolated had an apparent Mr of 59,000. Carbohydrate analysis as well as deglycosylation studies showed that the change in molecular weight was due to increased glycosylation.     

Purification and characterization of a bovine pregnancy-associated glycoprotein

A 67000 Mr bovine pregnancy-associated glycoprotein (bPAG) has been isolated from fetal cotyledons and purified to homogeneity by HPLC. The purification was monitored by a double immunodiffusion test and by RIA in conjunction with an antiserum raised against a crude fraction of placenta-specific antigens. The molecular weight of bPAG was estimated to be 67000 by SDS-PAGE. The isoelectric points (pI) of the four isoforms, determined by high-resolution analytical electrofocusing in polyacrylamide gel, were 4.4, 4.6, 5.2, and 5.4. The carbohydrate content of the bPAG consisted of approximately 10.02 +/- 1.09% neutral sugar and variant amounts of sialic acid (from 0.29 +/- 0.06% in the most basic isoform to 2.1 +/- 0.31% in the most acidic isoform). A specific antiserum was raised against the purified bPAG. A specific RIA showed that the bPAG was antigenically unrelated to BSA, alphafetoprotein (AFP), and human schwangerschafts-spezifischen (pregnancy-specific) beta 1 glycoprotein (SP1). According to some characteristics (e.g. the molecular weight), the purified bPAG may correspond to a form of the pregnancy-specific protein B previously described by Sasser and colleagues (Biol Reprod 1986; 35:936-942).     

Purification of Aeromonas hydrophila major outer-membrane proteins: N-terminal sequence analysis and channel-forming properties

Four outer-membrane proteins of Aeromonas hydrophila were purified and their N-terminal sequences and channel-forming properties were determined. Three could be matched with proteins from other species. One was a maltoporin, as its level increased when cells were grown in maltose-containing media, and the channel it formed was blocked by maltose. Another was like OmpF and OmpC of Escherichia coli, except that its channel fluctuated much more rapidly. The third protein, which was produced in low-phosphate medium, exhibited several properties of the general anion porin PhoE. The fourth showed no similarity to any known proteins. It had a unique N-terminus and it formed small sharply-defined cation-selective channels. Two other proteins which corresponded to OmpW of Vibrio cholerae and E. coli OmpA were partly characterized.     

Purification and characterization of a soybean leaf storage glycoprotein

Removing the pods from soybean (Glycine max [L.] Merr. cv Wye) plants induces a change in leaf function which is characterized by a change in the leaf soluble protein pattern. The synthesis of at least four polypeptides (~27, 29, 54, and 80 kilodaltons) is enhanced, and these polypeptides accumulate to levels comprising over 50% of the soluble protein. Heat girdling the petiole also causes the accumulation of these polypeptides, suggesting that the signal for changing leaf function may be associated with inhibition of phloem transport. The 27 and 29 kilodalton polypeptides are glycosylated and have been purified to greater than 90% by (NH₄)₂SO₄ fractionation, concanavilin A affinity, and gel filtration chromatography. These peptides appear to comprise a single protein. Mouse antiserum has been prepared against this glycoprotein and has been used to check for cross-reactivity with seed proteins and to quantitate changes with leaf development. No cross-reactivity was observed with seed soluble proteins from several stages of development. Quantitation showed the highest content in podded plants at, and shortly following, flowering, with levels subsequently declining in conjunction with seed growth. In depodded plants, the level of glycoprotein continued to increase following flowering and accounted for 45% of the soluble leaf protein by 4 weeks after depodding.     

Purification and distribution of a novel macrophage-specific calmodulin-binding glycoprotein

The murine macrophage-like cell line J774.16 and peritoneal exudate cells elicited with thioglycollate or starch contain a major calmodulin (CaM)-binding protein (CaMBP) which is absent in trifluoperazine-resistant variants of J774, resident peritoneal macrophages and peritoneal macrophages elicited with concanavalin A, lipopolysaccharide, proteose peptone, or Bacillus Calmette-Guerin. Resident murine peritoneal cells maintained in tissue culture for 3 days begin to accumulate this protein, as do human peripheral blood monocytes after 7 days of culture. A specific competitive displacement radioimmunoassay was developed with the use of a rabbit antiserum raised to the partially purified CaM-binding protein and [125I]CaM covalently cross-linked to the principal CaM-binding protein in the preparation. The radioimmunoassay confirmed the unique cellular distribution of this protein, suggesting that it may be a marker for certain stages of macrophage differentiation. Monoclonal antibodies were prepared, and one of these was used to further purify the protein by immunoaffinity chromatography. A protein of Mr 50,000 to 60,000 was isolated. The protein could be selectively adsorbed to wheat germ agglutinin agarose and subsequently eluted with N-acetyl glucosamine. This property, plus the sensitivity of the protein to endoglycosidase F, led to the conclusion that it is a glycoprotein. The cellular distribution, subcellular localization, and evidence for glycosylation suggest that this protein may be a macrophage-specific receptor with a high affinity for Ca2+-CaM.     

Purification and chemical study of a Collocalia glycoprotein]

A glycoprotein was purified from the aqueous extract of "edible bird's nest" (Collocalia) using free flow preparative electrophoresis and represented the main fraction of Collocalia glycoproteins. This glycoprotein is homogeneous upon agarose electrophoresis and slightly polydisperse upon ultracentrifugation (S So 20w = 3,0). The carbohydrate moiety contains galactose, mannose, glucosamine, galactosamine and sialic acid, which is completely released by Clostridium perfringens or Diplococcus pneumoniae neuraminidases and has the same chromatographic behaviour as N-acetyl-neuraminic acid. The peptide part of the glycoprotein is rich in serine, threonine and proline. About 40 p. cent of the hydroxyaminoacids are involved in carbohydrate-peptide linkages.     

Purification and N-terminal amino-acid sequence analysis of rat polymorphonuclear leukocyte cathepsin G

Cathepsin G was purified by single-step cation-exchange chromatography from rat polymorphonuclear leukocytes, obtained from the peritoneal cavity after induction of a mild peritonitis. The 26 N-terminal amino acids were determined and showed 73% identity to those of human cathepsin G. Total amino-acid composition demonstrated a high degree of basic amino acids in accordance with its high affinity for the cationic-exchange gel medium. The protein was found to be a glycoprotein with a glucosamine content of 7.4% of the calculated Mr28,900. On SDS/polyacrylamide-gel electrophoresis the protein showed a Mr of 28,400. It migrated as two bands in a gradient SDS/polyacrylamide-gel indicating isoforms. The pH optimum for the proteinase was determined to be 8.0-8.5 using Suc-Ala-Ala-Pro-Phe-Nan as substrate (Suc = 3-carboxypropionyl; Nan = 4-nitroanilide). Km and Kcat/Km values for Suc-Ala-Ala-Pro-Phe-Nan were 0.86mM and 280M-1S-1 and for Suc-Phe-Leu-Phe-Nan 0.24mM and 3600M-1S-1, respectively.     

The effect of partial deglycosylation on the structure of alpha1-acid glycoprotein

Changes in structure of alpha1-acid glycoprotein were followed after deglycosylation with neuraminidase, peptide N-glycohydrolase F or with a mixture of exoglycosidases. Partially deglycosylated preparations of alpha1-acid glycoprotein free of sialic acids, one complete saccharide component, sialic acids and one saccharide component and sialic acids and some of the external saccharides were obtained. The effect of these changes in saccharide components on the glycoprotein structure was studied by temperature perturbation difference spectroscopy, fluorescence spectroscopy, fourth-derivative of absorption spectra and spectra of CD. Partial deglycosylation resulted in transformation of the molecule to a more compact state in which phenylalanyl residues were even more buried, tyrosyl residues became more uniform and tryptophyl residues were less exposed. The content of ordered secondary structures decreased. The thermal stability of the molecule was not significantly affected. Removal of one of the five saccharide components from the native molecule had apparently deeper effect than total desialyzation of the glycoprotein.     

Purification and N-terminal analyses of algal biliproteins

1. R-, B- and C-phycoerythrins and R- and C-phycocyanins were isolated and purified on a preparative scale by calcium phosphate chromatography, ammonium sulphate fractionation and crystallization. 2. The N-terminal residues of these biliproteins were analysed. Methionine is the only N-terminal residue of all the phycoerythrins, there being about 14 N-terminal residues per molecule of R- and B-phycoerythrins (mol.wt. 290000) and about 8 per molecule of C-phycoerythrin (mol.wt. 226000). Threonine (1 residue) is N-terminal in C-phycocyanin (mol.wt. 138000), and both threonine (about 1·3 residues) and methionine (5 residues) are N-terminal in R-phycocyanin (mol.wt. 273000). 3. Results suggest that the apoproteins of the various phycoerythrins are closely related, whereas C-phycocyanin has quite a different gross structure, and that R-phycocyanin contains two types of sub-unit, one related to C-phycocyanin and the other to the phycoerythrins.     

Purification and characterization of a glycoprotein elicitor from Alternaria tenuissima

Glycoprotein elicitor can induce plant resistance and become a potential agent for biological control of plant diseases. Here, a new glycoprotein elicitor was purified with the method of cold alcohol precipitation and anion exchange chromatography from the mycelium of Alternaria tenuissima strain JH505, which was identified on the basis of morphological features and sequence analysis of rDNA internal transcribed spacer. The protein showed a single band on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) stained with silver and appeared one main protein peak in HPLC. The apparent molecular weight of the purified protein was 66 kDa and isoelectric point was about 4.27. This protein was identified as glycoprotein by glycoprotein staining Kit. Anthrone-colorimetric assay and Coomassie blue G-250 staining showed that carbohydrate and protein content was in a ratio of 1.75. After deglycosylation by trifluoromethane-sulfonic acid, this glycoprotein showed two bands on the SDS–PAGE, and which means the glycoprotein may have at least two glycosylation sites. The glycoprotein induced tobacco resistance against tobacco mosaic virus and enhanced wheat seedling growth at 15°C. The glycoprotein elicitor provided an effective way of alternative strategies for plant disease control.     

Partial enzymatic deglycosylation preserves the structure of cleaved recombinant HIV-1 envelope glycoprotein trimers

The trimeric envelope glycoprotein complex (Env) is the focus of vaccine development programs aimed at generating protective humoral responses to human immunodeficiency virus type 1 (HIV-1). N-Linked glycans, which constitute almost half of the molecular mass of the external Env domains, produce considerable structural heterogeneity and are a major impediment to crystallization studies. Moreover, by shielding the peptide backbone, glycans can block attempts to generate neutralizing antibodies against a substantial subset of potential epitopes when Env proteins are used as immunogens. Here, we describe the partial deglycosylation of soluble, cleaved recombinant Env trimers by inhibition of the synthesis of complex N-glycans during Env production, followed by treatment with glycosidases under conditions that preserve Env trimer integrity. The partially deglycosylated trimers are stable, and neither abnormally sensitive to proteolytic digestion nor prone to aggregation. Moreover, the deglycosylated trimers retain or increase their ability to bind CD4 and antibodies that are directed to conformational epitopes, including the CD4-binding site and the V3 region. However, as expected, they do not react with glycan-dependent antibodies 2G12 and PGT123, or the C-type lectin receptor DC-SIGN. Electron microscopic analysis shows that partially deglycosylated trimers have a structure similar to fully glycosylated trimers, indicating that removal of glycans does not substantially perturb the structural integrity of the trimer. The glycan-depleted Env trimers should be useful for structural and immunogenicity studies.     

Structural studies of a french bean storage protein: phaseolin

Molecular weights and sedimentation coefficients have been measured for different oligomeric forms of phaseolin, the major storage protein in seeds of Phaseolus vulgaris L. The results indicate that phaseolin is a trimer ($\text{M}{}_{\mathrm{r}}\text{= 150000}$) at neutral pH which aggregates further to a dodecamer form ($\text{M}{}_{\mathrm{r}}\text{= 596000}$) at pH 4.5. The subunit size is in good agreement with the recently determined sequence molecular weight, if allowance is made for bound oligosaccharide and phytic acid moieties. The trimeric nature at neutral pH has been confirmed by chemical crosslinking studies using dimethylsuberimidate and dithiobis(succinimidylpropionate). Analyses of optical rotatory dispersion and circular dichroism data have been used to examine the corformation of phaseolin. In common with other seed globulins, a low proportion of α-helix ($\text{～ 10\%}$) coupled with a high level of β-sheet ($\text{～50\%}$) is predicted. These data are compared with a structural analysis based on the amino acid sequence of a phaseolin subunit polypeptide. The predicted level of α-helix is increased ($\text{～20\%}$) when phaseolin is heated in sodium dodecyl sulphate, but not when the detergent is added at room temperature.     

The three-dimensional structure of the seed storage protein phaseolin at 3 A resolution.

The polypeptides of the trimeric seed storage protein phaseolin comprise two structurally similar units each made up of a beta-barrel and an alpha-helical domain. The beta-barrel has the 'jelly-roll' folding topology of the viral coat proteins and the alpha-helical domain shows structural similarity to the helix-turn-helix motif found in certain DNA-binding proteins.     

Purification and MALDI-MS characterization of stressin, a stress-associated glycoprotein.

Glycoconjugates have a whole spectrum of biological roles, from those that appear trivial to those that are crucial. Results accumulated in the past years indicate they might also play an important role in the response to stress, a complex physiological response of the human organism to various threats. We have recently identified stressin, a human serum glycoprotein, which was found to be increased under stress conditions. Here we report the purification of stressin from sera of professional soldiers and partial characterization of its protein and carbohydrate parts using lectin blotting and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Stressin was purified using a combination of ammonium sulfate precipitation, ion exchange chromatography, preparative gel electrophoresis and reverse-phase HPLC. It was found to be a highly glycosylated protein. Only 21.9 kDa (out of 36.7 kDa) was the protein part, whereas the remaining 40% of the mass originated from N-linked oligosaccharides. The carbohydrate part contained 12 sialic acids moieties, nearly 90% of which were lost due to post-source decay in the field-free tube. Tryptic fragments were produced from glycosylated and deglycosylated stressin, separated by reverse-phase HPLC and their exact molecular masses were determined using MALDI-MS. Comparison with tryptic maps of other proteins in computer databases indicated that stressin does not correspond to any already described protein.     

Purification and characterization of a sulfated glycoprotein secreted by Sertoli cells

Sulfated glycoprotein 2 (SGP-2), the major secretion product of Sertoli cells, was purified from cell culture medium by reverse-phase high-performance liquid chromatography. The native protein consists of disulfide-linked monomers of 41,000 and 29,000 daltons which have a strong tendency to associate into multimers. The purified SGP-2 was subjected to amino acid analysis and contained high levels of Asx (11.1%), Glx (15.1%), and leucine (11.5%). The oligosaccharides on the purified SGP-2 were analyzed to determine the monosaccharide compositions and the molecular weights of the intact carbohydrate moieties. SGP-2 was shown to be 23.7% carbohydrate and consisted of 1% fucose, 3.5% mannose, 4.1% galactose, 7.1% N-acetylglucosamine, and 8.0% N-acetylneuraminic acid. No N-acetylgalactosamine was detected. When the SGP-2 was digested with proteases, the intact oligosaccharides were chromatographed over a Bio-Gel P-6 column and found to elute in a single symmetrical peak of approximately 3,300 g/mol. On the basis of these results, the oligosaccharides on SGP-2 were proposed to consist of triantennary chains similar to those found on fetuin. When the 35SO4(2-)-labeled SGP-2 was digested with Pronase, the free amino acids could be separated by chromatography from the oligosaccharide. The 35SO4(2-) was shown to be associated with the oligosaccharide portion of SGP-2.     

The glycoprotein precursor of concanavalin A is converted to an active lectin by deglycosylation.

We have previously shown that concanavalin A is synthesized as a glycoprotein precursor that is unable to bind to sugars and is processed through six intermediate forms before assembly of the mature active lectin. Since processing involves removal of the N-glycan, four proteolytic steps and a religation, the precise event that leads to carbohydrate binding activity was not known. We have now purified the glycoprotein precursor from microsomal membranes and show that deglycosylation in vitro is sufficient alone to convert the precursor to an active carbohydrate binding protein. This is the first demonstration of a novel role for N-glycans and N-glycanases in the regulation of protein activity.