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Aberrant glycosylation and the overexpression of certain carbohydrate moieties is a consistent feature of cancers, and tumor-associated oligosaccharides are actively investigated as targets for immunotherapy. One of the most common aberrations in glycosylation patterns is the presentation of a single O-linkedN-acetylgalactosamine on a threonine or serine residue known as the “Tn antigen.” Whereas the ubiquitous nature of Tn antigens on cancers has made them a natural focus of vaccine research, such carbohydrate moieties are not always tumor-specific and have been observed on embryonic and nonmalignant adult tissue. Here we report the structural basis of binding of a complex of a monoclonal antibody (237mAb) with a truly tumor-specific glycopeptide containing the Tn antigen. In contrast to glycopeptide-specific antibodies in complex with simple peptides, 237mAb does not recognize a conformational epitope induced in the peptide by sugar substitution. Instead, 237mAb uses a pocket coded by germ-line genes to completely envelope the carbohydrate moiety itself while interacting with the peptide moiety in a shallow groove. Thus, 237mAb achieves its striking tumor specificity, with no observed physiological cross-reactivity to the unglycosylated peptide or the free glycan, by a combination of multiple weak but specific interactions to both the peptide and to the glycan portions of the antigen.

In order to explore monoclonal antibody recognition carbohydrate antigens, several structures from two monoclonal antibodies directed against carbohydrate epitopes derived from chlamydial LPS have been solved to high resolution. With the exception of CDR H3, antibodies S54-10 and S73-2 are both derived from the same set of germline gene segments as the previously reported structures S25-2 and S45-18. Despite this similarity, the antibodies differ in specificity and the mechanism by which they recognize their cognate antigen. S54-10 uses an unrelated CDR H3 to recognize its antigen in a fashion analogous to S45-18; however, S73-2 recognizes the same antigen as S45-18 and S54-10 in a wholly unrelated manner. Together, these antibody-antigen structures provide snapshots into how the immune system uses the same set of inherited germline gene segments to generate multiple possible specificities that allow for differential recognition of epitopes, and how unrelated CDR H3 sequences can result in convergent binding of clinically-relevant bacterial antigens.

High-resolution structures reveal how a germline antibody can recognize a range of clinically relevant carbohydrate epitopes. The germline response to a carbohydrate immunogen can be critical to survivability, with selection for antibody gene segments that both confer protection against common pathogens and retain the flexibility to adapt to new disease organisms. We show here that antibody S25-2 binds several distinct inner-core epitopes of bacterial lipopolysaccharides (LPSs) by linking an inherited monosaccharide residue binding site with a subset of complementarity-determining regions (CDRs) of limited flexibility positioned to recognize the remainder of an array of different epitopes. This strategy allows germline antibodies to adapt to different epitopes while minimizing entropic penalties associated with the immobilization of labile CDRs upon binding of antigen, and provides insight into the link between the genetic origin of individual CDRs and their respective roles in antigen recognition.

The histoblood-group ABO carbohydrate antigens are well known as important factors in blood transfusions, but they can also act as receptors for infectious agents and have been implicated in susceptibility to certain carcinomas. A single-chain variable-domain antigen-binding fragment (scFv) gene based on the known sequence of an anti-blood-group-A monoclonal antibody (AC1001) has been synthesized and expressed in Escherichia coli. The purified scFv preparation existed primarily in the monomeric form but also contained large amounts of dimeric and higher oligomeric forms. The corresponding variable-domain antigen-binding fragment (Fv) was generated by cleaving the VL–VH linker with subtilisin, and its activity was demonstrated by surface plasmon resonance with an immobilized bovine serum albumin A–trisaccharide conjugate (KD = 290 µM). AC1001 Fv crystals grown in the presence of N-acetylgalactosamine diffracted to 0.93 Å resolution. This is the first reported example of a crystal of an antibody antigen-binding fragment diffracting to atomic resolution.

Extracellular enzyme preparations from Streptomyces flavogriseus and Streptomyces olivochromogenes cultures grown on cellulose contained primarily cellulase activities, but similar preparations from cultures grown on xylan-containing materials possessed high levels of both cellulase and xylanase activities. Growth conditions that gave high endoxylanase levels also resulted in the production of enzymes involved in the hydrolysis of the nonxylose components of xylan. Specific acetyl xylan esterase activities were identified in enzyme preparations from both organisms. Both organisms also produced α- l -arabinofuranosidase activity that was not associated with endoxylanase activity. Other activities produced were α- l - O -methylglucuronidase and ferulic acid esterase. The latter enzyme was produced only by S. olivochromogenes and is an activity which has not previously been identified as a component of hemicellulase preparations.

Biodegradation of hemicelluloses requires enzyme activities that remove nonxylose substituents from the xylan backbone in addition to endoxylanases and β-xylosidases. Activities removing o-acetyl, arabinose, cinnamic acid-based esters, and uronic acid substituents occurred inStreptomyces olivochromogenes, Aspergillus niger, andSchizophyllum commune. 4-O-Methylglucuronidase was coinduced with other substituent-hydrolyzing enzymes when appropriate lignocellulosic materials were provided. Fractionation of crude enzymes indicated that the 4-o-methylglucuronidases were polydisperse. The 4-o-methylglucuronidases fromS. commune andA. niger acted on high molecular weight heteroxylans, but those from S.olivochromogenes functioned only with small, endo-β-(1,4)-xylanse-solubilized fragments.

Screening of some mesophilic species of Streptomyces indicated that enzyme activities capable of removing O-acetyl, arabinosyl, 4-O-methylglucuronyl, and feruloyl substituents from xylan were present in culture filtrates, in addition to endo-β-(1,4)-xylanases. Low constitutive levels of acetyl xylan esterase, arabinosidase, 4-O-methylglucuronidase, and ferulic acid esterase could be greatly elevated by supplementation of liquid media with starch-free wheat bran, oat spelts xylan, or sugar cane bagasse. One organism, S. olivochromogenes, produced particularly high levels of acetyl xylan esterase, ferulic acid esterase, arabinosidase, and 4-O-methylglucuronidase as well as endo-β-(1,4)-xylanase. Partial purification of extracellular preparations from S. flavogriseus and S. olivochromogenes disclosed the presence of multiple forms of both anionic and cationic enzyme activities. Several of the hydrolysing activities occurred as “families” in association with endo-β-(1,4)-xylanase.

A small-scale chemostat has been used to assess the basic nutritional requirements of curdlan producing strains of Alcaligenes faecalis. A lean, chemically-defined mineral salts medium has been formulated compatible with operation of a continuous culture at dilution rates equivalent to μxax observed in batch-culture. Molar growth yields for carbon, nitrogen, phosphorus and oxygen have been determined. The optimized minimal salts medium with 2.5% glucose and 90mM NH4Cl supported a steady-state nitrogen-limited biomass of 8.9g dry biomass/L at a max. dilution rate of 0.23hr−1. By virtue of its ability to increase μmax, the addition of yeast extract to the chemostat nutrient feed resulted in a significant increase in the max. biomass productivity of the continuous fermenter. Under these conditions curdlan is not synthesized in chemostat culture. Operation of a nitrogen-limited chemostat at Dmax resulted in the complete replacement of the original uracil-requiring mutant (ATCC 21680) by a non uracil-requiring variant strain of A. faecalis. Both the parent auxotroph and apparent revertant strain display similar physiological properties including the capacity to synthesize an extracellular, water-insoluble, neutral β-1,3-glucan polymer (curdlan) under appropriate non-growing conditions.

Three genes coding for xylanase synthesis in Clostridium thermocellum were cloned and expressed in Escherichia coli. Genomic DNA from Clostridium thermocellum was digested to completion with HindIII, BamHI, and SalI. The fragments were ligated into the corresponding sites of pUC19 and transformed into Escherichia coli. Two of the genes encoded for xylanases which depolymerized xylans but were unable to extensively convert these substrates to reducing sugar. The third gene encoded for an enzyme that extensively hydrolyzed xylan. The insert containing the latter gene was subjected to extensive mapping and was found to encode for a xylanase with a molecular weight of approximately 25,000. The protein product of the cloned gene was obtained in a relatively pure form by heat treatment, ion exchange and gel permeation steps. The enzyme was quite stable to high temperatures with a half-life of 24 h at 70 degrees C.

Two genes coding for xylanase synthesis in Bacillus circulans were cloned and expressed in Escherichia coli. After digestion of genomic DNA from Bacillus circulans with EcoRI and PstI, the fragments were ligated into the corresponding sites of pUC19 and transformed into Escherichia coli. Restriction enzyme mapping of the two inserts coding for xylanase activity indicated distinctly different nucleotide sequences. Cross-hybridization assays confirmed the absence of sequence homology between the two genes. In vitro transcription-translation assays indicated that the cloned genes encoded for proteins with molecular weights of 22,000 and 59,000. The gene products displayed different substrate specificities. The 22,000-dalton enzyme readily hybrolyzed aspeen, larchwood, and oat spelt xylans, whereas the second was unable to extensively depolymerize oat spelt xylan and resulted in very limited reducing sugar release from any of the xylan substrates tested. Both of the xylanases had isoelectric points of approximately 9.0.

Publisher Summary The present chapter describes chromatographic and electrophoretic techniques for the detection and measurement of α-(4- O -methyl)-D-glucuronidase and D-glucuronidase activities with specific reference to heteroxylans. The chapter tabulates the chromatographic properties of neutral and acidic monosaccharides and disaccharides. An indication that 4- O -methyl-D-glucuronic acid is the product of both snail and streptomycete enzyme action is provided, using both chromatographic columns, by the linear increase with incubation times of 30-120 min of the area of peaks with retention times for the 4- O -methylglucuronic acid derivative. Confirmation of the identity of the 6.5 and 2.6 min peaks as the appropriate silyl derivative is made by GC-mass spectrometry employing methane chemical ionization and electron impact. It has been reported that alkaline extraction of 4- O -methyl-Dglucurono-D-xylan may result in β-elimination of some uronyl moieties, generating an unsaturated hexuronate.

A 4.0-kilobase (kb) fragment of Bacillus circulans genomic DNA inserted into pUC19 and encoding endoxylanase activity was subjected to a series of subclonings. A 1.0-kb HindIII-HincII subfragment was found to code for xylanase activity. Maximum expression levels were observed with a subclone that contained an additional 0.3-kb sequence upstream from the coding region. Enhancer sequences in the upstream region are thought to be responsible for these high expression levels. Southern hybridization analyses revealed that the cloned gene hybridized with genomic DNA from Bacillus subtilis and Bacillus polymyxa. Xylanase activity expressed by Escherichia coli harboring the cloned gene was located primarily in the intracellular fraction. Levels of up to 7 U/ml or 35 mg/liter were obtained. The protein product was purified by ion exchange and gel permeation chromatography. The xylanase had a molecular weight of 20,500 and an isoelectric point of 9.0.

Publisher Summary Acetylxylan esterase occurs in a number of microorganisms and is known to act cooperatively with xylanases in the depolymerization of xylan. This particular enzyme activity has been determined either by release of acetic acid from partially purified preparations of acetylxylan from hardwoods or by measuring the release of 4-nitrophenol from 4-nitrophenyl acetate. As the lack of readily available sources of more natural substrates makes difficult the estimation of acetylxylan esterase activities, this chapter describes a method that has been developed to prepare chemically acetylated xylan from various commercial xylans. These materials, along with 4-nitrophenyl acetate, have been used for the measurement of acetylxylan esterase produced by a variety of Streptomyces species. The chapter discusses the estimation of acetic acid by high-performance liquid chromatography (HPLC).

Streptomyces flavogriseus CMCase and Avicelase were very stable at 30 degrees C but not at 40 degrees C or higher. beta-Glucosidase was less stable at all temperatures tested. Stabilities were similar at pH values between 5.5 and 7, the optimal range for enzyme activity. Cellulose solubilizing activity was reduced by 40% at a cellobiose concentration of 150 mM but glucose inhibited activity by only 10% at this concentration. beta-Glucosidase was inhibited by 40% at a glucose concentration of 10mM (ten times the substrate concentration). Relatively dilute S. flavogriseus cellulase extensively hydrolysed acid-swollen cellulose at concentrations as high as 10%. More highly crystalline forms of cellulose were more resistant to attack.

Viomycin resistance in a mutant of Rhizobium meliloti was found to involve an accumulation of phospholipid in the cell envelope. 14C-labelling revealed that this accumulation occurred at the expense: of neutral lipids in the envelope. Viomycin was shown to form complexes with phospholipid under artificial conditions and it is suggested that phospholipid-vionycin interactions also occur within the surface layers of the cell, resulting in a decreased permeability toward the antibiotic in the resistant cells.