A comparison of protein quantitation assays for biopharmaceutical applications.

Dye-based protein determination assays are widely used to estimate protein concentration, however various reports suggest that the response is dependent on the composition and sequence of the protein, limiting confidence in the resulting concentration estimates. In this study a diverse set of model proteins representing various sizes of protein and covalent modifications, some typical of biopharmaceuticals have been used to assess the utility of dye-based protein concentration assays. The protein concentration assays (Bicinchoninic acid (BCA), Bradford, 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA), DC, Fluorescamine and Quant-i) were compared to the 'gold standard' assay, quantitative amino acid analysis (AAA). The assays that displayed the lowest variability between proteins, BCA and DC, also generated improved estimates when BSA was used as a standard, when compared to AAA derived concentrations. Assays read out by absorbance tended to display enhanced robustness and repeatability, whereas the fluorescence based assays had wider quantitation ranges and lower limits of detection. Protein modification, in the form of glycosylation and PEGylation, and the addition of excipients, were found to affect the estimation of protein concentration for some of the assays when compared to the unmodified protein. We discuss the suitability and limitations of the selected assays for the estimation of protein concentration in biopharmaceutical applications.

The failure of different strains of Yersinia pestis to produce lipopolysaccharide O-antigen under different growth conditions is due to mutations in the O-antigen gene cluster.

The lipopolysaccharide (LPS) from eight strains of Yersinia pestis which had been cultured at 28 degrees C appeared to be devoid of an O-antigen when analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. LPS isolated from three of these strains which had been cultured at 37 degrees C also appeared to be devoid of an O-antigen. When the LPS from Y. pestis strain CO92 was purified and analysed by matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry, the observed signals were in the mass range predicted for molecules containing lipid A plus the core oligosaccharide but lacking an O-antigen. The nucleotide sequence of Y. pestis strain CO92 revealed the presence of a putative O-antigen gene cluster. However, frame-shift mutations in the ddhB, gmd, fcl and ushA genes are likely to prevent expression of the O-antigen thus explaining the loss of phenotype.

Characterization of the lipopolysaccharide of Yersinia pestis.

Lipopolysaccharide (LPS) extracted from eight strains of Yersinia pestis, which had been cultured at 28 or 37 degrees C, reacted equally well, in Western blots, with four monoclonal antibodies generated against the LPS from a single strain of Y. pestis cultured at 28 degrees C. LPS was extracted and purified from Y. pestis strain GB, which had been cultured at 28 degrees C. When the LPS was analysed by SDS-PAGE and MALDI-TOF mass spectrometry it was found to be devoid of an O-antigen. The LPS possessed activity of 2.7 endotoxin units/ng in the Limulus amoebocyte lysate assay. The LPS stimulated the production of TNFalpha and IL-6 from mouse macrophages, but was less active in these assays than LPS isolated from Escherichia coli strain 0111. Y. pestis LPS, either alone or with cholera toxin B subunit, was used to immunize mice. Either immunization schedule resulted in the development of an antibody response to LPS. However, this response did not provide protection against 100 MLD of Y. pestis strain GB.

Carbohydrate moieties in human secretory component.

Human secretory component has seven putative sites for N-linked glycosylation. From tryptic and Glu-C digests we have isolated peptides encompassing asparagines 65, 72, 117, 168, 403, 451 and 481. Analysis by on line HPLC-electrospray mass spectrometry indicated that these residues were fully glycosylated and that the major carbohydrate moieties were far less diversified in composition than expected. Fast atom bombardment mass spectrometry performed on oligosaccharides released by peptide-N-glycosidase F treatment of fractionated and unfractionated SC digests showed the following glycan compositions: Fuc(2)Hex(5)HexNAc(4), Fuc(3)Hex(5)HexNAc(4), NeuAcFucHex(5)HexNAc(4), NeuAcFuc(2)Hex(5)HexNAc(4), NeuAc(2)Hex(5)HexNAc4 and NeuAc(2)FucHex(5)HexNAc(4). Three of these oligosaccharides are the major carbohydrate moieties in human lactoferrin. A possible biological role of the secretory component glycans in the protection of mucosal surfaces is discussed.

Structural studies of N-glycans of filarial parasites. Conservation of phosphorylcholine-substituted glycans among species and discovery of novel chito-oligomers.

N-Type glycans containing phosphorylcholine (PC-glycans), unusual structures found in the important human pathogens filarial nematodes, represent a novel target for chemotherapy. Previous work in our laboratories produced compositional information on the PC-glycan of ES-62, a secreted protein of the rodent parasite Acanthocheilonema viteae. In particular, we established using fast atom bombardment mass spectrometry (MS) analysis that PC was attached to a glycan with a trimannosyl core, with and without core fucosylation, carrying between one and four additional N-acetylglucosamine residues. In the present study, we demonstrate that this structure is conserved among filarial nematodes, including the parasite of humans, Onchocerca volvulus, for which new drugs are most urgently sought. Furthermore, by employing a variety of procedures, including collision-activated dissociation MS-MS analysis and matrix-assisted laser desorption MS analysis, we reveal that surprisingly, filarial nematodes also contain N-linked glycans, the antennae of which are composed of chito-oligomers. To our knowledge, this is the first report of such structures in a eukaryotic glycoprotein.

alpha1-Antitrypsin Portland, a bioengineered serpin highly selective for furin: application as an antipathogenic agent.

The important role of furin in the proteolytic activation of many pathogenic molecules has made this endoprotease a target for the development of potent and selective antiproteolytic agents. Here, we demonstrate the utility of the protein-based inhibitor alpha1-antitrypsin Portland (alpha1-PDX) as an antipathogenic agent that can be used prophylactically to block furin-dependent cell killing by Pseudomonas exotoxin A. Biochemical analysis of the specificity of a bacterially expressed His- and FLAG-tagged alpha1-PDX (alpha1-PDX/hf) revealed the selectivity of the alpha1-PDX/hf reactive site loop for furin (Ki, 600 pM) but not for other proprotein convertase family members or other unrelated endoproteases. Kinetic studies show that alpha1-PDX/hf inhibits furin by a slow tight-binding mechanism characteristic of serpin molecules and functions as a suicide substrate inhibitor. Once bound to furin's active site, alpha1-PDX/hf partitions with equal probability to undergo proteolysis by furin at the C-terminal side of the reactive center -Arg355-Ile-Pro-Arg358- downward arrow or to form a kinetically trapped SDS-stable complex with the enzyme. This partitioning between the complex-forming and proteolytic pathways contributes to the ability of alpha1-PDX/hf to differentially inhibit members of the proprotein convertase family. Finally, we propose a structural model of the alpha1-PDX-reactive site loop that explains the high degree of enzyme selectivity of this serpin and which can be used to generate small molecule furin inhibitors.

Characterization of recombinant human plasma lecithin: cholesterol acyltransferase (LCAT): N-linked carbohydrate structures and catalytic properties.

The major N-linked carbohydrate structures were determined for recombinant human plasma lecithin:cholesterol acyltransferase (LCAT). The analysis of the structure of oligosaccharides by fast atom bombardment mass spectrometry (FAB-MS) and linkage analysis was preceded by reduction and carboxymethylation of the intact glycoproteins and digestion with trypsin and proline specific endopeptidase. The N-glycans were subsequently released from the glycopeptides by PNGase F digestion and the oligosaccharides were separated using a C18 Sep-pak cartridge. The data from the combination of FAB spectrometry and linkage analysis show that the N-linked glycans present on recombinant LCAT (rLCAT) were composed primarily of triantennary and tetraantennary structures with and without core fucosylation. A minor population of glycans (less than 5%) contained up to three repeats of N-acetyllactosamine in one or more antennae. The LCAT activities of both recombinant and circulating forms of plasma LCAT were determined using low molecular weight and lipoprotein substrates. The catalytic behavior of these two enzyme forms were found to be very similar if not identical. These findings validate the concept that the recombinant enzyme can serve as an appropriate model for structure/function studies of LCAT and provide the foundation for subsequent structural studies.

Human free secretory component is composed of the first 585 amino acid residues of the polymeric immunoglobulin receptor.

The main objective of this work was to unequivocally determine the C-terminal sequence of human milk free secretory component (SC). It was found to end at arginine-585, i.e. 33 amino acids downstream from the major heterogeneous C-terminal residue previously identified for colostrum SC. In contrast, our data showed that the C-terminal end of SC was found to be homogeneous. Conflicting assignments, Asp/Gln, a missing Asn-211, Asp/Asn, Glu/Gln were corrected and found to agree with the cDNA sequence. An Ala/Val substitution at position 562 (domain VI) was identified. Its genetic significance is uncertain at present.

A partial reductive-cleavage study of the capsular polysaccharide of Escherichia coli K57.

Trideuteriomethylated and methylated derivatives of the capsular polysaccharide of Escherichia coli K57 were partially cleaved by Et3SiH, using Me3SiOSO2 Me and Me3SiOSO2CF3 as catalysts, to produce oligosaccharide-anhydroalditols. The structures of the trideuteriomethylated trisaccharide- and tetrasaccharide-anhydroalditols isolated were established by FABMS and NMR spectroscopy. Although conditions for the selective production of the tetrasaccharide-anhydroalditol could not be established, oligosaccharide-anhydroalditols were isolated in sufficiently high yield to make this an attractive approach for the structural elucidation of the repeating units of bacterial polysaccharides.

Novel tyvelose-containing tri- and tetra-antennary N-glycans in the immunodominant antigens of the intracellular parasite Trichinella spiralis.

The larval stage of the intestinal nematode, Trichinella spiralis, secretes and displays on its cuticle a number of antigenically cross-reactive glycoproteins. These so-called TSL-1 antigens induce a powerful antibody response in parasitized animals. In rats, anti-TSL-1 antibodies mediate a protective immunity that expels invading larvae from the intestine. The vast majority of anti-TSL-1 antibodies are specific for glycans. Although the biological functions of TSL-1 antigens are not known, the powerful effect of glycan-specific antibodies on the intestinal survival of T. spiralis suggests that they play an important role in parasite establishment. Little is known about the structures of the glycans present on the TSL-1 glycoproteins. Recent studies have suggested, however, that the antigens contain very unusual glycans (Wisnewski, N., McNeil, M., Grieve, R.B. and Wassom, D.L., Mol. Biochem. Parasitol., 61, 25-36, 1993). Sugar and linkage analysis of the combined secreted products unexpectedly showed that a major terminal sugar is tyvelose (3,6-dideoxy-D-arabino-hexose; Tyv) which has previously been found only in certain gram-negative bacterial lipopolysaccharides. In this paper, we report the first rigorous structural study of oligosaccharides released from TSL-1 antigens by peptide N-glycosidase F digestion. Using strategies based on fast atom bombardment mass spectrometry (FAB-MS), we have discovered a novel family of tri- and tetra-antennary N-glycans whose antennae are comprised of the tyvelose-capped structure: Tyv1,3GalNAc beta 1,4(Fuc alpha 1,3)GlcNAc beta 1-. Thus a major population of TSL-1 glycans contains clusters of hydrophobic terminal structures which are likely to be highly immunogenic.

Glycans as targets for monoclonal antibodies that protect rats against Trichinella spiralis.

We have investigated the role of glycans on Trichinella spiralis antigens in recognition by rat monoclonal antibodies (mAbs) which protect rat pups against challenge with the parasite. In pups born to infected dams or pups passively immunized with mAbs, antibodies eliminate a challenge dose from the intestine within hours ('rapid expulsion'). Because such dramatic protection can be afforded by mAbs, we have sought to characterize the parasite antigens they target. In this report we show that protective antibodies were unable to bind excretory/secretory (ES) antigens deglycosylated with trifluoromethanesulphonic acid (TFMS). In addition, oligosaccharides isolated from glycoproteins by alkaline hydrolysis or peptide: N glycosidase F (PNGase F) digestion were bound by protective, but not non-protective, mAbs. Glycans affinity purified with protective mAb 9D bound to all but one protective mAb. These antibodies have been shown previously to bind to the surfaces of intact larvae, indicating that the glycan is exposed on the parasite surface. Candidate glycans that may be involved in binding protective mAbs have unusual tri- and tetra-antennary structures with terminal tyvelose moieties (Reason et al., Glycobiology, 4, 000-000, 1994). Coating of the larval surface with such glycans may serve to protect the parasite and its secreted products from enzymatic attack as the parasite travels to and resides in its epithelial niche.

Identification and oligosaccharide structure analysis of rhodopsin glycoforms containing galactose and sialic acid.

The N-linked oligosaccharides of frog (Rana pipiens) rhodopsin were analysed by sequential exoglycosidase digestion and gel filtration chromatography, following reductive tritiation. In addition, selected tryptic glycopeptides obtained from frog retinal rod outer segment membranes were examined by electrospray mass spectrometry (ES-MS), fast atom bombardment mass spectrometry (FAB-MS), amino acid sequence and composition analysis, and carbohydrate composition analysis. The amino acid sequence data demonstrated that the glycopeptides were derived from rhodopsin and confirmed the presence of two N-glycosylation sites, at residues Asn2 and Asn15. The predominant glycan (approximately 60% of total) had the structure GlcNAc beta 1-2Man alpha 1-3(Man alpha 1-6) Man beta 1-4GlcNAc beta 1-4GlcNAc-(Asn), with the remaining structures containing 1-3 additional hexose residues, as reported previously for bovine rhodopsin. Unlike bovine rhodopsin, however, a sizable fraction of the total glycans of frog rhodopsin also contained sialic acid (NeuAc), with the sialylated oligosaccharides being present exclusively at the Asn2 site. FAB-MS analysis of oligosaccharides released from the Asn2 site gave, among other signals, an abundant quasimolecular ion corresponding to a glycan of composition NeuAc1Hex6HexNAc3 (where Hex is hexose and HexNAc is N-acetylhexosamine), consistent with a hybrid structure. The potential biological implications of these results are discussed in the context of rod outer segment membrane renewal.

Carbohydrate analysis.

Carbohydrate analysis is an active field that is expanding rapidly. Hundreds of new structures are reported each year and methods for screening glycopolymers for known structures are now becoming accessible to the nonspecialist. Detailed structure analysis of recombinant glycoproteins has become relatively routine in specialist laboratories. Rapid advances are being made in the understanding of structure and function of biologically active carbohydrates that are of interest to the pharmaceutical industry.

Localization of O-GlcNAc modification on the serum response transcription factor.

A unique form of nucleoplasmic and cytoplasmic protein glycosylation, O-linked GlcNAc, has previously been detected, using Gal transferase labeling techniques, on a myriad of proteins (for review see Hart, G. W., Haltiwanger, R. S., Holt, G. D., and Kelly, W. G. (1989a) Annu. Rev. Biochem. 58, 841-874), including many RNA polymerase II transcription factors (Jackson, S. P., and Tjian, R. (1988) Cell 55, 125-133). However, virtually nothing is known about the degree of glycosylation at individual sites, or, indeed, the actual sites of attachment of O-GlcNAc on transcription factors. In this paper we provide rigorous evidence for the occurrence and locations of O-GlcNAc on the c-fos transcription factor, serum response factor (SRF), expressed in an insect cell line. Fast atom bombardment mass spectrometry (FAB-MS) of proteolytic digests of SRF provides evidence for the presence of a single substoichiometric O-GlcNAc residue on each of four peptides isolated after sequential cyanogen bromide, tryptic, and proline specific enzyme digestion: these peptides are 306VSASVSP312, 274GTTSTIQTAP283, 313SAVSSADGTVLK324, and 374DSSTDLTQTSSSGTVTLP391. Using an array of techniques, including manual Edman degradation, aminopeptidase, and elastase digestion, together with FAB-MS, the major sites of O-GlcNAc attachment were shown to be serine residues within short tandem repeat regions. The highest level of glycosylation was found on the SSS tandem repeat of peptide (374-391) which is situated within the transcriptional activation domain of SRF. The other glycosylation sites observed in SRF are located in the region of the protein between the DNA binding domain and the transcriptional activation domain. Glycosylation of peptides (274-283) and (313-324) was found to occur on the serine in the TTST tandem repeat and on serine 316 in the SS repeat, respectively. The lowest level of glycosylation was recovered in peptide (306-312) which lacks tandem repeats. All the glycosylation sites identified in SRF are situated in a relatively short region of the primary sequence close to or within the transcriptional activation domain which is distant from the major sites of phosphorylation catalyzed by casein kinase II.

Vertebrate lens alpha-crystallins are modified by O-linked N-acetylglucosamine.

Crystallins are structural proteins responsible for establishing the remarkable optical properties of the lens. Yet many of these highly conserved proteins are also expressed in nonocular tissues, where they have alternative functions apparently unrelated to their structural role in the lens. Here we report that lens alpha-crystallins, some of which function as heat-shock proteins in other tissues, are modified with O-linked N-acetylglucosamine (O-GlcNAc). An in vitro enzymatic assay that transfers [3H]Gal to terminal GlcNAc moieties labels alpha A and alpha B crystallins in lens homogenates from man, rhesus monkey, rat, cow, and rhea (an ostrich-like bird). O-Linkage of the saccharide is demonstrated by sensitivity to base-catalyzed beta-elimination and resistance to peptide:N-glycosidase F treatment. Chromatographic analyses of the beta-elimination products and fast atom bombardment-mass spectrometry of [3H]Gal-labeled tryptic peptides confirm the saccharide structure. Isoelectric focusing of [3H]Gal-labeled bovine lens proteins reveals the presence of O-GlcNAc on all four alpha-crystallin subunits, A1, A2, B1, and B2. Electrospray mass spectrometry of bovine alpha-crystallin demonstrates the presence of a single O-GlcNAc substitution on alpha A2. Gas-phase protein sequencing and fast atom bombardment-mass spectrometry of the major radiolabeled tryptic peptide from bovine alpha-crystallin reveal that GlcNAc is attached to the alpha A subunits at serine 162. This post-translational modification may play an important role in the molecular organization of lens alpha-crystallin.

Characterisation of the N-linked oligosaccharides of the light chain of human glycoprotein IIb by f.a.b.-m.s.

The glycosylation of the light chain (GPIIbL) of glycoprotein IIb, one of the glycoproteins constituting the receptor for fibrinogen, fibronectin, and the von Willebrand factor on platelet cell surfaces, was investigated using fast-atom-bombardment mass spectrometry (f.a.b.-m.s.). Complex-type N-glycans were observed, attached to Asn-60. The most abundant oligosaccharide is a disialylated biantennary structure substituted with fucose on the chitobiose core. Mono-sialylated biantennary, and di- and tri-sialylated triantennary structures were found as minor constituents of the N-glycan population. The amino acid sequence of GPIIbL was fully mapped by f.a.b.-m.s., thereby providing the first direct evidence for the absence of O-glycosylation.

High-sensitivity FAB-MS strategies for O-GlcNAc characterization.

In this paper we report the first application of fast atom bombardment mass spectrometry (FAB-MS) to O-linked N-acetylglucosamine (O-GlcNAc)-bearing glycopeptides. Using N-acetylgalactosamine (GalNAc)- and Gal-GalNAc-containing glycopeptides (isolated from Tn glycophorin and desialylated normal glycophorin, respectively) as readily available model compounds, rapid and sensitive derivatization/FAB-MS strategies applicable to serine/threonine-rich glycopeptides have been devised. Peptides and glycopeptides were propionylated in a 1 min reaction using a mixture of trifluoroacetic anhydride and propionic acid, and the product mixtures were analysed directly by FAB-MS. Glycopeptides and peptides rich in hydroxylated residues afforded characteristic clusters of molecular ions at high sensitivity. Additional sensitivity enhancement was achieved by prior esterification of carboxyl groups. These methods were used in a study of O-GlcNAc glycopeptides produced by purified O-GlcNAc transferase addition of GlcNAc to the synthetic peptides YSDSPSTST and YSGSPSTST in which Y is tyrosine, S is serine, D is aspartic acid, P is proline, T is threonine and G is glycine. The propionyl derivatives afforded high-quality spectra which unequivocally showed that the majority of the glycopeptides were substituted with a single GlcNAc residue. Low pmol quantities of material gave detectable signals. The propionylation/FAB-MS procedure has been combined with gas-phase sequencing strategies and shows promise for defining the sites of glycosylation of O-GlcNAc glycopeptides that are available in limited quantities.