Monoclonal antibody recognizes a conformational epitope in a random coil protein

Saad, Bashar; Corradin, G; Bosshard, Hans Rudolf

doi:10.1111/j.1432-1033.1988.tb14446.x

Cited by 19 publications

(9 citation statements)

References 28 publications

(12 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Rather, the THRGP CD spectra resemble that of denatured collagen and fall into the 'catch-all' category of unordered conformation (34). the poorly defined term random coil which clearly does not preclude the THRGP secondary structure deduced here (34), or the presence of secondary structure in other random coil proteins; for example, there is even a report of a monoclonal antibody that can recognize a conformational epitope in a random coil protein (27). At this point, there is sufficient structural and chemical similarity with the extensins (15,16) to consider the THRGP as an analogous protein, although its lack of polyproline-II helix might argue against homology, were it not for the following sequencing data.…”

Section: Discussionmentioning

confidence: 99%

Structure of the Threonine-Rich Extensin from Zea mays

Kieliszewski¹,

Leykam²,

Lamport³

1990

Plant Physiol.

View full text Add to dashboard Cite

Chymotryptic digestion of a threonine-rich hydroxyproline-rich glycoprotein (THRGP) purified from the cell surface of a Zea mays cell suspension culture gave a peptide map dominated by the hexadecapeptide TC5: Thr-Hyp-Ser-Hyp-Lys-Pro-Hyp-Thr-ProLys-Pro-Thr-Hyp-Hyp-Thr-Tyr, in which the repetitive motif SerHyp-Lys-Pro-Hyp-Thr-Pro-Lys is homologous with the dominant decamer of P1-type dicot extensins: Ser-Hyp-Hyp-Hyp-Hyp-ThrHyp-Val-Tyr-Lys, modified by a Lys for Hyp substitution at residue 3, a Val-Tyr deletion at residues 8 and 9, and incomplete posttranslational modification of proline residues. One of the minor peptides (TC1) contained the 8-residue sequence: Thr-Hyp-SerHyp-Hyp-Hyp-Hyp-Tyr corresponding to the C-terminal tail (judging from the recently isolated maize cDNA clone MC56) which is homologous with the major repetitive motif of the 'P3' class of dicot extensins. Direct peptide sequencing defined potential glycosylated regions on the THRGP corresponding to clone MC56 and showing that glycosylated and nonglycosylated domains altemate with high regularity. The THRGP is not in the polyproline-11 conformation, judging from circular dichroic spectra, but nevertheless is an extended rod, from electron microscopic data. HFsolvolysis of cell walls from maize coleoptile, root, and root tip released deglycosylated THRGP detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblots with high titer rabbit polyclonal antibodies raised against the intact THRGP. In a quantitative enzyme-linked immunosorbent assay, these antibodies cross-reacted 20% with tomato P1 extensin, and 18% with anhydrous hydrogen fluoride-deglycosylated P1. These results, together with other previously published data, show that maize THRGP is homologous with the dicot P1 extensins and, as such, is the first extensin isolated from a graminaceous monocot.In 1987, we reported (15) 2 Abbreviations: THRGP, threonine-hydroxyproline-rich glycoprotein; HRGP, hydroxyproline-rich glycoprotein; dTHRGP, deglycosylated threonine-hydroxyproline-rich glycoproteins; dw, dry weight; P1, glycosylated tomato extensin type 1; dPI, deglycosylated tomato extensin type P1; P2, glycosylated tomato extensin type 2; dP2, deglycosylated tomato extensin type 2; HHRGP, histidinehydroxyproline-rich glycoprotein; dHHRGP, deglycosylated histidine-hydroxyproline-rich glycoprotein; HF, anhydrous hydrogen fluoride; OPA, orthophthalaldehyde; HFBA, hepta-fluorobutyric acid; AGP, arabinogalactan protein; AP, alkaline phosphatase. CD, circular dichroism; ABTS, 2,2'-Azino-bis(3-ethylbenzthiazinoline-6-sulfonic acid).ceous monocot Zea mays and provided evidence that this HRGP may be an extensin, a class ofextended rodlike HRGPs well known as structural components of the dicot primary cell wall. Like a typical extensin, this protein is highly basic, rich in hydroxyproline and proline, 0-glycosylated through hydroxyproline residues with one to four arabinosyl residues, and was visualized by transmission EM as a flexuous rod. Furthermore, antibodies raised ...

show abstract

Section: Discussionmentioning

confidence: 99%

Structure of the Threonine-Rich Extensin from Zea mays

Kieliszewski¹,

Leykam²,

Lamport³

1990

Plant Physiol.

View full text Add to dashboard Cite

show abstract

“…Cytochrome c was type I11 from Sigma and was further purified as described (Saad et al, 1988). Biotinylated cytochrome c was prepared by reaction with the Nhydroxysuccinimide ester of biotin (Fluka) as before (Ngai et al, 1993).…”

Section: Methodsmentioning

confidence: 99%

Real‐Time Monitoring of Antigen‐antibody Recognition on a Metal Oxide Surface by an Optical Grating Coupler Sensor

Bernard

Bosshard

1995

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

Real-time monitoring of intermolecular interactions can provide a direct and rapid estimate of the affinity and kinetics of interactions between biomolecules. Optical methods based on the measurement of changes of refractive index in the immediate vicinity of a liquid-solid interface are particularly convenient because they require no radioactive, fluorescent or other labelling of the molecules under study. In the present work we have followed the specific interaction of protein molecules on a SiO,/TiO, surface with the help of the optical grating coupler sensor instrument BIOS-1. This instrument allows the determination of the absolute mass of protein adsorbed to the sensor surface and, therefore, the calculation of the molar ratio of the components partaking in an intermolecular interaction. For example, about 3 ng avidirdmm' surface area could be adsorbed. This amount closely corresponds to a monolayer composed of densely packed globular avidin molecules. A dimeric, biotinylated leucine zipper peptide was bound to this avidin layer at a molar ratio of 1 : 1 (1 peptide molecule/4 biotin binding sites of tetrameric avidin). An average of 1J2.6 peptides was recognized by a peptide-specific monoclonal antibody. Even though avidin was not covalently bound to the sensor surface, the avidin-coated chip could be used repeatedly to measure the time course of antibody binding as a function of the concentration of the antibody. From such measurements it was possible to calculate the association and dissociation rate constants assuming that the interaction of the antibody with the surface-bound antigen can be described by a simple Langmuir binding model. The limits of the Langmuir model are discussed. The same antigen-antibody reaction was also analyzed by a surface plasmon resonance biosensor (BIAcoreTM, Pharmacia). The results obtained with the two instruments, which register different optical phenomena and employ different surface chemistry, were in good agreement.

show abstract

“…These were performed on 96-well microtiter plates as described before in more detail [14]. Briefly, plates were coated with 1 pg cytochrome c2, 20 pM cytochrome c, peptides or apo-cytochrome c2, in 50 pl buffer A for 1 h at 37 C, or overnight at 4°C.…”

Section: Elisamentioning

confidence: 99%

Antigenic sites on cytochrome c₂ from Rhodospirillum rubrum

Saad

Bosshard

1990

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

The antigenic determinants for three monoclonal antibodies against cytochrome c2 from Rhodospirillum vuhrum were partially characterized by differential chemical modification of free and antibody-bound cytochrome c2 and by cross-reactivity analysis with different antigens. Circular dichroism spectroscopy was used to probe the effect of antibody binding on the conformation of cytochrome c 2 . The binding of two antibodies was strongly dependent on the native folding of the antigen. The first antibody bound to a determinant around the exposed heme edge on the 'front side' of the molecule which is not antigenic in mitochondrial cytochrome c2. Binding of this antibody to cytochrome c increased the induced CD of the ferric heme in a manner similar to that observed previously when mitochondrial cytochrome-c oxidase bound to the front side of cytochrome c. This observation points to a subtle conformational adaptation of the antigen induced by the antibody. The determinant for the second antibody, which also affected the heme CD spectrum of the antigen, was on a polypeptide loop where cytochrome c2 differs from mitochondrial cytochrome c by an eight-residue insertion. The third antibody, which did not induce a change in CD, bound to a sequential determinant near the amino end of cytochrome c2. Only this antibody cross-reacted with isolated cytochrome-c-derived peptides and with apo-cytochrome c2.

show abstract

Monoclonal antibody recognizes a conformational epitope in a random coil protein

Cited by 19 publications

References 28 publications

Structure of the Threonine-Rich Extensin from Zea mays

Structure of the Threonine-Rich Extensin from Zea mays

Real‐Time Monitoring of Antigen‐antibody Recognition on a Metal Oxide Surface by an Optical Grating Coupler Sensor

Antigenic sites on cytochrome c₂ from Rhodospirillum rubrum

Contact Info

Product

Resources

About

Monoclonal antibody recognizes a conformational epitope in a random coil protein

Cited by 19 publications

References 28 publications

Structure of the Threonine-Rich Extensin from Zea mays

Structure of the Threonine-Rich Extensin from Zea mays

Real‐Time Monitoring of Antigen‐antibody Recognition on a Metal Oxide Surface by an Optical Grating Coupler Sensor

Antigenic sites on cytochrome c2 from Rhodospirillum rubrum

Contact Info

Product

Resources

About

Antigenic sites on cytochrome c₂ from Rhodospirillum rubrum