The roles of the N-linked glycans and extension regions of soybean beta-conglycinin in folding, assembly and structural features

Beta-conglycinin, one of the dominant storage proteins of soybean, is a trimer composed of three subunits, alpha, alpha' and beta. All subunits are N-glycosylated and alpha and alpha' contain extension regions in addition to the core regions common to all subunits. Non-glycosylated individual subunits and deletion mutants (alpha(c) and alpha'(c)) lacking the extension regions of alpha and alpha' were expressed in Escherichia coli. All recombinant proteins were purified to near homogeneity and appeared to have the correct conformation, as judged by CD, density-gradient centrifugation and gel-filtration profiles, indicating that the N-linked glycans and extension regions are not essential for the folding and the assembly into trimers of beta-conglycinin. Density-gradient centrifugation, gel-filtration and differential scanning calorimetry profiles of the recombinant proteins and the native beta-conglycinin indicated that the N-linked glycans and extension regions contribute to the dimension of beta-conglycinin but not to the density and the thermal stability. Comparing the solubilities of the individual subunits with those of deletion mutants, only the alpha and alpha' subunits were soluble at lower ionic strength (mu < 0.25) at around the pH value of the endoplasmic reticulum. This suggests that the extension regions play an important role in the prevention of aggregation in the endoplasmic reticulum in analogy with the N-linked glycans.     

Domain-switched mouse IgM/IgG2b hybrids indicate individual roles for C mu 2, C mu 3, and C mu 4 domains in the regulation of the interaction of IgM with complement C1q

Although polymeric IgM and monomeric IgG are potent activators of the classical complement pathway, previous studies have indicated that monomeric IgM is inactive. To understand this and to examine the roles of the individual mu domains in complement activation, we created a set of IgM/IgG2b mouse chimeric Abs in which homologous domains of both Abs have been interchanged, either singly or together with adjacent domains. The monomer subunits (H2L2) of the resulting chimeras were analyzed for their capacities to bind C1q and to initiate complement-mediated lysis (CML) of haptenated erythrocytes. When C gamma 2 was flanked by C mu 4, the inherent C1q-binding activity of the C gamma 2 domain was lost. This demonstrates that C mu 4 can suppress the C1q-binding activity of the adjacent C gamma 2 domain, and suggests that C mu 4 may exert a similar effect on the C mu 3 domain in the IgM monomer subunit. When C mu 3 was located in an IgG2b background and potentially freed from the constraints imposed by the IgM background, the monomer was not able to bind C1q or initiate CML. This suggests that these activities are not expressed inherently in the C mu 3 domain. The transplantation of C mu 3 together with C mu 4 into the IgG background permitted polymer formation. This polymer was able to bind C1q, although neither the monomer nor the polymer forms were active in CML; conversely, all IgM polymers with a transplanted C gamma 2 domain were active in both C1q binding and CML, and demonstrated apparent Kd values similar to that of wild-type IgM.     

Crystallization of a deglycosylated T cell receptor (TCR) complexed with an anti-TCR Fab fragment

A strategy to overexpress T cell receptors (TCRs) in Lec3.2.8.1 cells has been developed using the "Velcro" leucine zipper sequence to facilitate alpha-beta pairing. Upon secretion in culture media, the VSV-8-specific/H2-Kb-restricted N15 TCR could be readily immunopurified using the anti-leucine zipper monoclonal antibody 2H11, with a yield of 5-10 mg/liter. Mass spectrometry analysis revealed that all attached glycans were GlcNAc2-Man5. Following Superdex 200 gel filtration to remove aggregates, wild-type N15 or N15(s), a C183S variant lacking the unpaired cysteine at amino acid residue 183 in the Cbeta domain, was thrombin-cleaved and endoglycosidase H-digested, and the two derivatives were termed iN15DeltaH and N15(s)DeltaH, respectively, and sized by Superdex 75 chromatography to high purity. N-terminal and C-terminal microsequencing analysis showed the expected unique termini of N15 alpha and beta subunits. Nevertheless, neither protein crystallized under a wide range of conditions. Subsequently, we produced a Fab fragment of the murine TCR Cbeta-specific hamster monoclonal antibody H57 and complexed the Fab fragment with iN15DeltaH and N15(s)DeltaH. Both N15(s)DeltaH-Fab[H57] and iN15DeltaH-Fab[H57] complexes crystallize, with the former diffracting to 2.8-A resolution. These findings show that neither intact glycans nor the conserved and partially exposed Cys-183 is required for protein stability. Furthermore, our results suggest that the H57 Fab fragment aids in the crystallization of TCRs by altering their molecular surface and/or stabilizing inherent conformational mobility.     

Dimerization of beta B2-crystallin: the role of the linker peptide and the N- and C-terminal extensions

beta B2- and gamma B-crystallins of vertebrate eye lens are 2-domain proteins in which each domain consists of 2 Greek key motifs connected by a linker peptide. Although the folding topologies of beta B2- and gamma B-domains are very similar, gamma B-crystallin is always monomeric, whereas beta B2-crystallin associates to homodimers. It has been suggested that the linker or the protruding N- and C-terminal arms of beta B2-crystallin (not present in gamma B) are a necessary requirement for this association. In order to investigate the role of these segments for dimerization, we constructed two beta B2 mutants. In the first mutant, the linker peptide was replaced with the one from gamma B (beta B2 gamma L). In the second mutant, the N- and C-terminal arms of 15- and 12-residues length were deleted (beta B2 delta NC). The beta B2 gamma L mutant is monomeric, whereas the beta B2 delta NC mutant forms dimers and tetramers that cannot be interconverted without denaturation. The spectral properties of the beta B2 mutants, as well as their stabilities against denaturants, resemble those of wild-type beta B2-crystallin, thus indicating that the overall peptide fold of the subunits is not changed significantly. We conclude that the peptide linker in beta B2-crystallin is necessary for dimerization, whereas the N- and C-terminal arms appear to be involved in preventing the formation of higher homo-oligomers.     

The medium subunits of adaptor complexes recognize distinct but overlapping sets of tyrosine-based sorting signals

Tyrosine-based sorting signals conforming to the motif YXXO (Y is tyrosine, X is any amino acid, and O is an amino acid with a bulky hydrophobic side chain (leucine, isoleucine, phenylalanine, methionine, valine)) interact with the medium (mu) subunits of clathrin adaptor (AP) complexes. We have analyzed the selectivity of interaction between YXXO signals and the mu1, mu2, and mu3 (A or B) subunits of the AP-1, AP-2, and AP-3 complexes, respectively, by screening a combinatorial XXXYXXO library using the yeast two-hybrid system. All the medium subunits were found to prefer proline at position Y+2, suggesting that YXXO signals are stabilized by a bend in the polypeptide backbone. Other than for this common preference, each medium subunit favored specific sets of residues at the X and O positions; these preferences were consistent with the proposed roles of the different adaptor complexes in rapid endocytosis and lysosomal targeting. A considerable specificity overlap was also revealed by these analyses, suggesting that additional factors, such as the context of the signals, must be important determinants of recognition.     

The hypervariable domain of the murine leukemia virus surface protein tolerates large insertions and deletions, enabling development of a retroviral particle display system

The surface proteins (SU) of murine type-C retroviruses have a central hypervariable domain devoid of cysteine and rich in proline. This 41-amino-acid region of Friend ecotropic murine leukemia virus SU was shown to be highly tolerant of insertions and deletions. Viruses in which either the N-terminal 30 amino acids or the C-terminal 22 amino acids of this region were replaced by the 7-amino-acid sequence ASAVAGA were fully infectious. Insertions of this 7-amino-acid sequence at the N terminus, center, and the C terminus of the hypervariable domain had little effect on envelope protein (Env) function, while this insertion at a position 10 amino acids following the N terminus partially destabilized the association between the SU and transmembrane subunits of Env. Large, complex domains (either a 252-amino-acid single-chain antibody binding domain [scFv] or a 96-amino-acid V1/V2 domain of HIV-1 SU containing eight N-linked glycosylation sites and two disulfides) did not interfere with Env function when inserted in the center or C-terminal portions of the hypervariable domain. The scFv domain inserted into the C-terminal region of the hypervariable domain was shown to mediate binding of antigen to viral particles, demonstrating that it folded into the active conformation and was displayed on the surface of the virion. Both positive and negative enrichment of virions expressing the V1/V2 sequence were achieved by using a monoclonal antibody specific for a conformational epitope presented by the inserted sequence. These results indicated that the hypervariable domain of Friend ecotropic SU does not contain any specific sequence or structure that is essential for Env function and demonstrated that insertions into this domain can be used to extend particle display methodologies to complex protein domains that require expression in eukaryotic cells for glycosylation and proper folding.     

Regulation of NMDA receptor phosphorylation by alternative splicing of the C-terminal domain

The NMDA (N-methyl D-aspartate) receptors in the brain play a critical role in synaptic plasticity, synaptogenesis and excitotoxicity. Molecular cloning has demonstrated that NMDA receptors consist of several homologous subunits (NMDAR1, 2A-2D). A variety of studies have suggested that protein phosphorylation of NMDA receptors may regulate their function and play a role in many forms of synaptic plasticity such as long-term potentiation. We have examined the phosphorylation of the NMDA receptor subunit NMDAR1 (NR1) by protein kinase C (PKC) in cells transiently expressing recombinant NR1 and in primary cultures of cortical neurons. PKC phosphorylation occurs on several distinct sites on the NR1 subunit. Most of these sites are contained within a single alternatively spliced exon in the C-terminal domain, which has previously been proposed to be on the extracellular side of the membrane. These results demonstrate that alternative splicing of the NR1 messenger RNA regulates its phosphorylation by PKC, and that mRNA splicing is a novel mechanism for regulating the sensitivity of glutamate receptors to protein phosphorylation. These results also provide evidence that the C-terminal domain of the NR1 protein is located intracellularly, suggesting that the proposed transmembrane topology model for glutamate receptors may be incorrect.     

NMR spectroscopy of alpha-crystallin. Insights into the structure, interactions and chaperone action of small heat-shock proteins

The subunit molecular mass of alpha-crystallin, like many small heat-shock proteins (sHsps), is around 20 kDa although the protein exists as a large aggregate of average mass around 800 kDa. Despite this large size, a well-resolved 1H NMR spectrum is observed for alpha-crystallin which arises from short, polar, highly-flexible and solvent-exposed C-terminal extensions in each of the subunits, alpha A- and alpha B-crystallin. These extensions are not involved in interactions with other proteins (e.g. beta- and gamma-crystallins) under non-chaperone conditions. As determined by NMR studies on mutants of alpha A-crystallin with alterations in its C-terminal extension, the extensions have an important role in acting as solubilising agents for the relatively-hydrophobic alpha-crystallin molecule and the high-molecular-weight (HMW) complex that forms during the chaperone action. The related sHsp, Hsp25, also exhibits a flexible C-terminal extension. Under chaperone conditions, and in the HMW complex isolated from old lenses, the C-terminal extension of the alpha A-crystallin subunit maintains its flexibility whereas the alpha B-crystallin subunit loses, at least partially, its flexibility, implying that it is involved in interaction with the 'substrate' protein. The conformation of 'substrate' proteins when they interact with alpha-crystallin has been probed by 1H NMR spectroscopy and it is concluded that alpha-crystallin interacts with 'substrate' proteins that are in a disordered molten globule state, but only when this state is on its way to large-scale aggregation and precipitation. By monitoring the 1H and 31P NMR spectra of alpha-crystallin in the presence of increasing concentrations of urea, it is proposed that alpha-crystallin adopts a two-domain structure with the larger C-terminal domain unfolding first in the presence of denaturant. All these data have been combined into a model for the quaternary structure of alpha-crystallin. The model has two layers each of approximately 40 subunits arranged in an annulus or toroid. A large central cavity is present whose entrance is ringed by the flexible C-terminal extensions. A large hydrophobic region in the aggregate is exposed to solution and is available for interaction with 'substrate' proteins during the chaperone action.     

Serological analysis of C-terminal region of alpha antigen from Mycobacterium avium-intracellulare complex and Mycobacterium tuberculosis

The alpha antigen, which is a 30 kDa protein secreted by mycobacterial species, is an immunodominant antigen. The C-terminal regions of alpha antigens are highly divergent, though there are regions where the amino acid sequence of alpha antigen is conserved. We investigated whether the C-terminal regions of the Mycobacterium avium alpha antigen, M. intracellulare alpha antigen and M. tuberculosis alpha antigen contain sequence-specific B-cell epitopes. The C-terminal regions of M. avium alpha antigen and M. intracelluare alpha antigen reacted to anti-M. avium alpha antigen but not to anti-M. tuberculosis alpha antigen derived from rabbits. Thus, M. avium and M. intracellulare have an antigenic determinant in common with rabbit. The C-terminal region of M. tuberculosis alpha antigen did not react to anti-M. avium alpha antigen or anti-M. tuberculosis alpha antigen. An enzyme-linked immunosorbent assay revealed that only the C-terminal region of M. avium alpha antigen reacted to the sera of two of six patients with M. avium-intracellulare (MAC) but not to the sera of patients with M. tuberculosis. In contrast, the C-terminal regions of M. intracellulare alpha antigen and M. tuberculosis alpha antigen were not recognized by the sera from patients with MAC or M. tuberculosis. This region of M. avium alpha antigen can produce a sequence-specific B-cell epitope in humans.     

Does the application of ground force set the energetic cost of cross-country skiing?

We tested whether the rate at which force is applied to the ground sets metabolic rates during classical-style roller skiing in four ways: 1) by increasing speed (from 2.5 to 4.5 m/s) during skiing with arms only, 2) by increasing speed (from 2.5 to 4.5 m/s) during skiing with legs only, 3) by changing stride rate (from 25 to 75 strides/min) at each of three speeds (3.0, 3.5, and 4.0 m/s) during skiing with legs only, and 4) by skiing with arms and legs together at three speeds (2.0-3.2 m/s, 1.5 degrees incline). We determined net metabolic rates from rates of O2 consumption (gross O2 consumption - standing O2 consumption) and rates of force application from the inverse period of pole-ground contact [1/tp(arms)] for the arms and the inverse period of propulsion [1/tp(legs)] for the legs. During arm-and-leg skiing at different speeds, metabolic rates changed in direct proportion to rates of force application, while the net ground force to counteract friction and gravity (F) was constant. Consequently, metabolic rates were described by a simple equation (metab = F . 1/tp . C, where metab is metabolic rates) with cost coefficients (C) of 8.2 and 0.16 J/N for arms and legs, respectively. Metabolic rates predicted from net ground forces and rates of force application during combined arm-and-leg skiing agreed with measured metabolic rates within +/-3. 5%. We conclude that rates of ground force application to support the weight of the body and overcome friction set the energetic cost of skiing and that the rate at which muscles expend metabolic energy during weight-bearing locomotion depends on the time course of their activation.     

Structural and functional analysis of J chain-deficient IgM

Previous studies have discerned two forms of polymeric mouse IgM: moderately cytolytic (complement-activating) pentamer, which contains J chain, and highly cytolytic hexamer, which lacks J chain. To investigate the relationships among polymeric structure, J chain content, and cytolytic activity, we produced IgM in J chain-deficient and J chain-proficient mouse hybridoma cell lines. Both hexamer and pentamer were produced in the absence as well as the presence of J chain. Hexameric IgM activated (guinea pig) complement approximately 100-fold more efficiently than did J chain-deficient pentamer, which, in turn, was more active than J chain-containing pentamer. These results are consistent with the hypothesis that J chain-containing pentamer cannot activate complement. We also analyzed the structure of IgM-S337, in which the mu-chain bears the C337S substitution. Like normal IgM, IgM-S337 was formed as a hexamer and as both J chain deficient- and J chain-containing pentamers. Unlike normal IgM, IgM-S337 dissociated in SDS into various subunits. For IgM-S337 pentamer, the predominant subunits migrated as mu2kappa2 and mu4kappa4, and the subunit distribution was unaltered by J chain. However, J chain was found only in the mu2kappa2 species, suggesting that some arrangement of inter-mu bonds directs incorporation of J chain. IgM-S337 hexamer also dissociated to mu2kappa2 and mu4kappa4, but also yielded several species migrating much more slowly in SDS-PAGE than wild-type mu12kappa12. To account for these forms, we propose that each mu-chain can interact with three other mu-chains and that some hexameric molecules contain two catenated mu6kappa6 circles.     

In vivo injection of antisense oligodeoxynucleotides to G alpha subunits and supraspinal analgesia evoked by mu and delta opioid agonists

For 5 consecutive days repeated intracerebroventricular (i.c.v.) administration of antisense oligodeoxynucleotides (ODNs) to G alpha subunit mRNAs was used to impair the function of mouse Gi1, Gi2, Gi3 and Gx/z regulatory proteins. Decreases of 20 to 60% on the G alpha-like immunoreactivity could be observed in neural structures of mouse brain, an effect that was not produced by a random-sequence ODN used as a control. The ODN to Gi1 alpha subunits lacked effect on opioid-evoked analgesia. In mice injected with the ODN to Gi2 alpha subunits the antinociceptive activity of all the opioids studied appeared greatly impaired. The ODN to Gi3 alpha subunits reduced the effects of the selective agonists of delta opioid receptors, [D-Pen2,5]-enkephalin and [D-Ala2]deltorphin II. Conversely, the analgesia evoked by opioids binding mu opioid receptors, [D-Ala2, N-MePhe4,Gly-ol5]enkephalin and morphine, appeared consistently and significantly attenuated in mice injected with the ODN to Gx/z alpha. The effect of the neuropeptide beta-endorphine-(1-31) agonist at mu and delta receptors was also reduced by ODNs to Gi3 alpha or Gx/z alpha subunits. l.c.v. injection of antibodies directed to these G alpha subunits antagonized opioid-induced analgesia with a pattern similar to that observed for the ODNs. Thus, the mu and delta opiod receptors regulate different classes of G transducer proteins to mediate the analgesic effect of agonists. The in vivo antisense strategy and the use of specific antibodies to G alpha subunits gave comparable results, indicating that in the neural tissue the mRNAs and the G alpha subunits can be accessed by the corresponding ODNs and IgGs.     

The development of anti-CD79 monoclonal antibodies for treatment of B-cell neoplastic disease

The B-cell antigen receptor (BCR) consists of cell surface IgM associated with the CD79 alpha/beta heterodimer. In this paper we describe a panel of monoclonal antibodies (mAbs) recognising the extracellular regions of human CD79 alpha and beta. FACS analysis demonstrated that the mAbs bind to a range of Burkitt's lymphoma lines, a mouse B-cell line (JO-72) transfected with human CD79 alpha and beta, and tumour biopsies from NHL patients. The specificity of the mAbs was confirmed by immunoprecipitation. The Ka for the binding of IgG from the anti-CD79 alpha mAbs to cell surface CD79 alpha on Ramos cells was 3 x 10(8) M-1, and their maximum level of binding, 1.7-2 x 10(5) molecules/cell, matched that obtained with anti-Fc mu and anti-Fd mu mAbs. All four anti-CD79 beta mAbs were of lower affinity. Interestingly, in growth arrest studies, we found that while all anti-Fc mu mAbs caused profound inhibition of proliferation of Ramos cells, a range of other anti-BCR mAbs, which included the anti-CD79, anti-Fab mu, anti-gamma and anti-idiotype reagents, all performed poorly giving a maximum of 25% inhibition. These differences in performance are believed to relate to the ability of anti-BCR mAbs to cross-link neighbouring surface BCR and suggest that, unlike anti-Fc mu which favours cross-linking, most of these mAbs are binding in a monogamous, non-cross-linking, union with the BCR.     

Mutational analysis of the N-linked glycans on Autographa californica nucleopolyhedrovirus gp64

gp64 is the major envelope glycoprotein in the budded form of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). gp64 is essential for AcMNPV infection, as it mediates penetration of budded virus into host cells via the endocytic pathway. In this study, we used site-directed mutagenesis to map the positions of the N-linked glycans on AcMNPV gp64, characterize their structures, and evaluate their influence on gp64 function. We found that four of the five consensus N-glycosylation sites in gp64 are used, and we mapped the positions of those sites to amino acids 198, 355, 385, and 426 in the polypeptide chain. Endoglycosidase H sensitivity assays showed that N-linked glycans located at different positions are processed to various degrees. Lectin blotting analyses showed that each N-linked glycan on gp64 contains alpha-linked mannose, all but one contains alpha-linked fucose, and none contains detectable beta-linked galactose or alpha2,6-linked sialic acid. The amounts of infectious progeny produced by AcMNPV mutants lacking one, two, or three N-linked glycans on gp64 were about 10- to 100-fold lower than wild-type levels. This reduction did not correlate with reductions in the expression, transport, or inherent fusogenic activity of the mutant gp64s or in the gp64 content of mutant budded virus particles. However, all of the mutant viruses bound more slowly than the wild type. Therefore, elimination of one or more N-glycosylation sites in AcMNPV gp64 impairs binding of budded virus to the cell, which explains why viruses containing these mutant forms of gp64 produce less infectious progeny.     

Antigen binding forces of individually addressed single-chain Fv antibody molecules

Antibody single-chain Fv fragment (scFv) molecules that are specific for fluorescein have been engineered with a C-terminal cysteine for a directed immobilization on a flat gold surface. Individual scFv molecules can be identified by atomic force microscopy. For selected molecules the antigen binding forces are then determined by using a tip modified with covalently immobilized antigen. An scFv mutant of 12% lower free energy for ligand binding exhibits a statistically significant 20% lower binding force. This strategy of covalent immobilization and measuring well separated single molecules allows the characterization of ligand binding forces in molecular repertoires at the single molecule level and will provide a deeper insight into biorecognition processes.     

A C-terminal helicase domain of the human papillomavirus E1 protein binds E2 and the DNA polymerase alpha-primase p68 subunit

The human papillomavirus (HPV) E1 and E2 proteins bind cooperatively to the viral origin of replication (ori), forming an E1-E2-ori complex that is essential for initiation of DNA replication. All other replication proteins, including DNA polymerase alpha-primase (polalpha-primase), are derived from the host cell. We have carried out a detailed analysis of the interactions of HPV type 16 (HPV-16) E1 with E2, ori, and the four polalpha-primase subunits. Deletion analysis showed that a C-terminal region of E1 (amino acids [aa] 432 to 583 or 617) is required for E2 binding. HPV-16 E1 was unable to bind the ori in the absence of E2, but the same C-terminal domain of E1 was sufficient to tether E1 to the ori via E2. Of the polalpha-primase subunits, only p68 bound E1, and binding was competitive with E2. The E1 region required (aa 397 to 583) was the same as that required for E2 binding but additionally contained 34 N-terminal residues. In confirmation of these differences, we found that a monoclonal antibody, mapping adjacent to the N-terminal junction of the p68-binding region, blocked E1-p68 but not E1-E2 binding. Sequence alignments and secondary-structure prediction for HPV-16 E1 and other superfamily 3 (SF3) viral helicases closely parallel the mapping data in suggesting that aa 439 to 623 constitute a discrete helicase domain. Assuming a common nucleoside triphosphate-binding fold, we have generated a structural model of this domain based on the X-ray structures of the hepatitis C virus and Bacillus stearothermophilus (SF2) helicases. The modelling closely matches the deletion analysis in suggesting that this region of E1 is indeed a structural domain, and our results suggest that it is multifunctional and critical to several stages of HPV DNA replication.     

Garlic (Allium sativum) lectins bind to high mannose oligosaccharide chains

Two mannose-binding lectins, Allium sativum agglutinin (ASA) I (25 kDa) and ASAIII (48 kDa), from garlic bulbs have been purified by affinity chromatography followed by gel filtration. The subunit structures of these lectins are different, but they display similar sugar specificities. Both ASAI and ASAIII are made up of 12.5- and 11.5-kDa subunits. In addition, a complex (136 kDa) comprising a polypeptide chain of 54 +/- 4 kDa and the subunits of ASAI and ASAIII elutes earlier than these lectins on gel filtration. The 54-kDa subunit is proven to be alliinase, which is known to form a complex with garlic lectins. Constituent subunits of ASAI and ASAIII exhibit the same sequence at their amino termini. ASAI and ASAIII recognize monosaccharides in mannosyl configuration. The potencies of the ligands for ASAs increase in the following order: mannobiose (Manalpha1-3Man) < mannotriose (Manalpha1-6Manalpha1-3Man) approximately mannopentaose < Man9-oligosaccharide. The addition of two GlcNAc residues at the reducing end of mannotriose or mannopentaose enhances their potencies significantly, whereas substitution of both alpha1-3- and alpha1-6-mannosyl residues of mannotriose with GlcNAc at the nonreducing end increases their activity only marginally. The best manno-oligosaccharide ligand is Man9GlcNAc2Asn, which bears several alpha1-2-linked mannose residues. Interaction with glycoproteins suggests that these lectins recognize internal mannose as well as bind to the core pentasaccharide of N-linked glycans even when it is sialylated. The strongest inhibitors are the high mannose-containing glycoproteins, which carry larger glycan chains. Indeed, invertase, which contains 85% of its mannose residues in species larger than Man20GlcNAc, exhibited the highest binding affinity. No other mannose- or mannose/glucose-binding lectin has been shown to display such a specificity.     

Identification of a novel AMP-activated protein kinase beta subunit isoform that is highly expressed in skeletal muscle

The AMP-activated protein kinase (AMPK) is a member of a growing family of related kinases, including the SNF1 complex in yeast, which respond to nutritional stress. AMPK is a heterotrimeric complex of a catalytic subunit (alpha) and two regulatory subunits (beta and gamma), and proteins related to all three subunits have been identified in the SNF1 complex. We have used the two-hybrid system in order to identify proteins interacting with the catalytic subunit (alpha2). Using this approach, we have isolated a novel AMPKbeta isoform, which we designate AMPKbeta2. The N-terminal region of beta2 differs significantly from that of the previously characterized isoform (beta1), suggesting that this region could play a role in isoform-specific AMPK activity. Comparison of the C-terminal sequences of beta1 and beta2 with their related proteins in yeast identifies two highly conserved regions predicted to be involved in binding of the alpha and gamma subunits. The expression of beta1 and beta2 was examined in a number of tissues, revealing that the beta1 isoform is highly expressed in liver with low expression in skeletal muscle, whereas the opposite pattern is observed for the beta2 isoform. These results suggest that the beta isoforms have tissue-specific roles, which may involve altered responses to upstream signaling and/or downstream targeting of the AMPK complex.