Kinesin's Light Chains Inhibit the Head- and Microtubule-Binding Activity of its Tail

Evidence was obtained that both the WA and BLA crossidiotype (XId) groups are conformational antigens requiring both L and H chains and that with heat denaturation the antigens that define the XIds and antigen-binding activity are lost in parallel. In contrast, the primary structure-dependent crossreactive idiotype (CRI), PSL2, which is only weakly detected on native Wa and Bla monoclonal rheumatoid factors (mRFs), became prominently detected on the heated Wa and Bla mRFs. Heat denaturation may provide a simple method for distinguishing Ids determined by conformational antigen from primary structure-dependent Ids. In addition to heat denaturation, some acid conditions commonly used for preparation of RFs were also found to cause marked loss of Id antigen. The finding of PSL2-CRI on Bla mRF indicates that this Id is not unique to the WA XId.

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Influence of Ser/Pro-rich domain and kinase domain of double cortin-like protein kinase on microtubule-binding activity

The Journal of Biochemistry ◽

10.1093/jb/mvr013 ◽

2011 ◽

Vol 149 (5) ◽

pp. 619-627 ◽

Cited By ~ 5

Author(s):

T. Nagamine ◽

S. Shimomura ◽

N. Sueyoshi ◽

I. Kameshita

Keyword(s):

Protein Kinase ◽

Kinase Domain ◽

Binding Activity ◽

Microtubule Binding ◽

Rich Domain

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SPECIFICITY IN THE COMBINATION OF FD FRAGMENTS WITH L CHAINS TO FORM HAPTEN-BINDING SITES

Journal of Experimental Medicine ◽

10.1084/jem.123.5.921 ◽

1966 ◽

Vol 123 (5) ◽

pp. 921-934 ◽

Cited By ~ 12

Author(s):

O. A. Roholt ◽

G. Radzimski ◽

D. Pressman

Keyword(s):

Amino Acid ◽

Propionic Acid ◽

Binding Sites ◽

Amino Acid Analysis ◽

Binding Activity ◽

Light Chains ◽

Antibody Activity ◽

Fc Fragment ◽

Fab Fragments ◽

Correct Alignment

In the work reported here we have shown that light chains and Fd fragments can be separated completely in propionic acid and then recombined to form Fab fragments with antibody activity. This experiment indicates that in the recombination a correct alignment of the Fd fragments and the L chains occurs to give a competent antibody site, just as occurs with the recombination of separated heavy and light chains of the antibody; thus the Fc fragment is not required for correct alignment. Fd fragments of antibody alone show very low binding activity toward the specific hapten. As is the case for the combination of heavy and light chains, the combination of Fd fragments and light chains also requires that both components come from antibody from the same rabbit in order to give binding sites. When they are derived from different rabbits producing antibody against the same antigen, they still give Fab fragments as shown by immunoelectrophoresis but do not have competent binding sites. An important observation is that the subunits of the papain digest fractions, FabI and FabII, have the capacity to cross-combine to form active Fab fragments with competent binding sites. FdI from FabI combines with LII chains from FabII to give the composite (FdI-LII) with good binding activity. Likewise, the composite (FdII-LI) has good binding activity. The composites from the two types of antibody molecules yielding different Fab fragments have antibody activity although heretofore these molecules have appeared to be different on the bases of chromatography and amino acid analysis. There is also a preferential combination of the Fd fragments to combine with the correct L fragments to give binding sites since this combination takes preference over the combination of Fd fragments of antibody with light chains of normal globulin (or of light chains of antibody with Fd fragments of normal globulin).

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The kinetoplastid kinetochore protein KKT4 is an unconventional microtubule tip–coupling protein

The Journal of Cell Biology ◽

10.1083/jcb.201711181 ◽

2018 ◽

Vol 217 (11) ◽

pp. 3886-3900 ◽

Cited By ~ 12

Author(s):

Aida Llauró ◽

Hanako Hayashi ◽

Megan E. Bailey ◽

Alex Wilson ◽

Patryk Ludzia ◽

...

Keyword(s):

Chromosome Segregation ◽

Binding Activity ◽

Significant Similarity ◽

Load Bearing ◽

Kinetochore Protein ◽

Microtubule Binding ◽

Binding Domains ◽

Microtubule Binding Domain ◽

Coupling Protein

Kinetochores are multiprotein machines that drive chromosome segregation by maintaining persistent, load-bearing linkages between chromosomes and dynamic microtubule tips. Kinetochores in commonly studied eukaryotes bind microtubules through widely conserved components like the Ndc80 complex. However, in evolutionarily divergent kinetoplastid species such as Trypanosoma brucei, which causes sleeping sickness, the kinetochores assemble from a unique set of proteins lacking homology to any known microtubule-binding domains. Here, we show that the T. brucei kinetochore protein KKT4 binds directly to microtubules and maintains load-bearing attachments to both growing and shortening microtubule tips. The protein localizes both to kinetochores and to spindle microtubules in vivo, and its depletion causes defects in chromosome segregation. We define a microtubule-binding domain within KKT4 and identify several charged residues important for its microtubule-binding activity. Thus, despite its lack of significant similarity to other known microtubule-binding proteins, KKT4 has key functions required for driving chromosome segregation. We propose that it represents a primary element of the kinetochore–microtubule interface in kinetoplastids.

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Expression of functional eGFP-fused antigen-binding fragment of ranibizumab in Pichia pastoris

Bioimpacts ◽

10.34172/bi.2021.23219 ◽

2021 ◽

Author(s):

Shirin Movaghar Asareh ◽

Tahereh Savei ◽

Sareh Arjmand ◽

Seyed Omid Ranaei Siadat ◽

Fataneh Fatemi ◽

...

Keyword(s):

Pichia Pastoris ◽

Fluorescent Protein ◽

Antibody Fragment ◽

Binding Activity ◽

Age Related Macular Degeneration ◽

Light Chains ◽

Antigen Binding ◽

Active Structure ◽

Age Related ◽

Fragment Antigen Binding

Introduction: Ranibizumab is a mouse monoclonal antibody fragment antigen-binding (Fab) against human vascular endothelial growth factor-A (VEGF-A), inhibiting angiogenesis. This antibody is commercially produced in Escherichia coli host and used to treat wet age-related macular degeneration (AMD).Methods: In this study, the heavy and light chains of ranibizumab were expressed in Pichia pastoris. The expressed chains were incubated overnight at 4°C for interaction. The formation of an active structure was evaluated based on the interaction with substrate VEGF-A using an indirect ELISA, and an electrochemical setup. Furthermore, reconstruction of split enhanced green fluorescent protein (eGFP) reporter, chimerized at the C-terminus of the heavy and light chains, was used to characterize chains’ interaction. Results: P. pastoris efficiently expressed designed constructs and secreted them into the culture medium. The anti-Fab antibody detected the constructed Fab structure in western blot analysis. Reconstruction of the split reporter confirmed the interaction between heavy and light chains. The designed ELISA and electrochemical setup results verified the binding activity of the recombinant Fab structure against VEGF-A. Conclusion: In this work, we indicated that the heavy and light chains of ranibizumab Fab fragments (with or without linkage to split parts of eGFP protein) were produced in P. pastoris. The fluorescence of reconstructed eGFP was detected after incubating the equal ratio of chimeric-heavy and light chains. Immunoassay and electrochemical tests verified the bioactivity of constructed Fab. The data suggested that P. pastoris could be considered a potential efficient eukaryotic host for ranibizumab production.

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Identification of a novel microtubule binding and assembly domain in the developmentally regulated inter-repeat region of tau

The Journal of Cell Biology ◽

10.1083/jcb.124.5.769 ◽

1994 ◽

Vol 124 (5) ◽

pp. 769-782 ◽

Cited By ~ 257

Author(s):

BL Goode ◽

SC Feinstein

Keyword(s):

Amino Acid ◽

Electrostatic Interactions ◽

Weak Interactions ◽

Binding Activity ◽

Site Directed Mutagenesis ◽

Dependent Manner ◽

Microtubule Organization ◽

Developmentally Regulated ◽

Microtubule Binding ◽

Amino Acid Repeats

Tau is a developmentally regulated microtubule-associated protein that influences microtubule behavior by directly associating with tubulin. The carboxyl terminus of tau contains multiple 18-amino acid repeats that bind microtubules and are separated by 13-14-amino acid inter-repeat (IR) regions previously thought to function as "linkers." Here, we have performed a high resolution deletion analysis of tau and identified the IR region located between repeats 1 and 2 (the R1-R2 IR) as a unique microtubule binding site with more than twice the binding affinity of any individual repeat. Truncation analyses and site-directed mutagenesis reveal that the binding activity of this site is derived primarily from lys265 and lys272, with a lesser contribution from lys271. These results predict strong, discrete electrostatic interactions between the R1-R2 IR and tubulin, in contrast to the distributed array of weak interactions thought to underlie the association between 18-amino acid repeats and microtubules (Butner, K. A., and M. W. Kirschner. J. Cell Biol. 115:717-730). Moreover, competition assays suggest that the R1-R2 IR associates with microtubules at tubulin site(s) distinct from those bound by the repeats. Finally, a synthetic peptide corresponding to just 10 amino acids of the R1-R2 IR is sufficient to promote tubulin polymerization in a sequence-dependent manner. Since the R1-R2 IR is specifically expressed in adult tau, its action may underlie some of the developmental transitions observed in neuronal microtubule organization. We suggest that the R1-R2 IR may establish an adult-specific, high affinity anchor that tethers the otherwise mobile tau molecule to the tubulin lattice, thereby increasing microtubule stability. Moreover, the absence of R1-R2 IR expression during early development may allow for the cytoskeletal plasticity required of immature neurons.

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A membrane-bound form of glutamate dehydrogenase possesses an ATP-dependent high-affinity microtubule-binding activity

Biochemical Journal ◽

10.1042/bj2950447 ◽

1993 ◽

Vol 295 (2) ◽

pp. 447-455 ◽

Cited By ~ 20

Author(s):

F Rajas ◽

B Rousset

Keyword(s):

Membrane Protein ◽

Glutamate Dehydrogenase ◽

Rat Liver ◽

Binding Activity ◽

Binding Studies ◽

Microtubule Binding ◽

Phase Partitioning ◽

Membrane Bound ◽

Commercial Sources

We previously identified a 50 kDa membrane protein which bound to in vitro assembled microtubules [Mithieux and Rousset (1989) J. Biol. Chem. 264, 4664-4668]. This protein exhibited the expected properties for mediating the ATP-dependent association of vesicles with microtubules [Mithieux, Audebet and Rousset (1988) Biochim. Biophys. Acta 969, 121-130]. The 50 kDa membrane protein (MP50), initially extracted in very low amount from isolated pig thyroid lysosomes/endosomes, has now been purified from membrane preparations of crude vesicle fractions from pig liver and brain. MP50 was isolated from detergent-solubilized membrane protein by affinity chromatography on immobilized ATP; 3-5 mg of MP50 was obtained from 100 g of liver tissue. Phase partitioning in Triton X-114 indicated that MP50 is a peripheral membrane protein. Radioiodinated liver MP50 bound to microtubules assembled in vitro. The binding was inhibited by ATP (Ki = 0.76 mM) and displaced by unlabelled liver or brain MP50. Equilibrium binding studies yielded KD values of 1.8 x 10(-7) M. By N-terminal amino acid sequence analysis, MP50 was identified as glutamate dehydrogenase (GDH), by comparison of V8 protease peptide maps of MP50 with purified liver GDH. Liver MP50 exhibited a low GDH activity; 4-5 units/mg compared with 18 and 34 units/mg for purified bovine and rat liver GDH respectively. Bovine and rat liver GDH yielded six spots from pI 5.7 to 7.2 when analysed by two-dimensional electrophoresis; in contrast, MP50 gave one main spot (corresponding to spot 2 of liver GDH) with a pI of approx. 6.5. Soluble liver GDH from commercial sources exhibited a very low or no microtubule-binding activity. In conclusion, we have found a membrane-bound form of GDH capable of specific and nucleotide-sensitive interaction with microtubules. Our data suggest that GDH isoproteins, the number of which has been undervalued up to now, could have cellular functions other than that of an enzyme.

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