Effect of enzymatic desialylation of human serum amyloid P component on surface exposure of laser photo CIDNP (chemically induced dynamic nuclear polarization) ‐ reactive histidine, tryptophan and tyrosine residues

Abstract

The human pentraxin serum amyioid P component (SAP) exhibits no microheterogeneity in its complex di-antennary glycan. To elucidate whether the removal of sialic acids from this glycoprotein might affect the accessibility of certain amino acid residues of the protein we employed the laser photo CIDNP approach as a sensitive tool. The CIDNP effect is created by the interaction of a photoexcited dye with reactive amino acids and results in enhanced absorption-or emission-signals which can be observed for the three aromatic amino acids histidine, tryptophan, and tyrosine if they are accessible to the dye. Therefore, this technique can be applied to explore surface exposure of these residues. The respective spectra of SAP and enzymatically desialylated SAP were determined. Six tryptophan/histidine signals and one tyrosine signal are present in the aromatic part of the CIDNP difference spectrum of SAP. The corresponding spectrum of desialylated SAP shows remarkable alterations. The chemical shift of one tryptophan/histidine-characteristic signal is decreased by 0.1 ppm. One tryptophan/histidine signal disappeared and a new one was formed in the CIDNP difference spectrum of desialylated SAP, while the other signals were unaffected. The tyrosine signal has a clearly enhanced intensity in desialylated SAP. Therefore, the removal of sialic acid moieties from the single N-glycan of each monomer apparently affects surface presentation of distinct ClDNP-reactive amino acids of SAP.Key words: Chemically induced dynamic nuclear polarization (CIDNP); Glycoprotein; Human serum amyloid P component (SAP); Sialic acid 3-sulfated derivatives of galactose, N-acetylgalactosamine and glucuronic acid, which are thought to influence the deposition of SAP to amyloid fibrils and bacterial surfaces [9][10][11][12]. The protein part of human SAP and its tissue form shown no disease-associated alterations, which is also true for threir glycosylation pattern [12]. SAP contains one invariant complex diantennary glycan Asn 3z, located on a fl-strand under the single c~-helix on the promoter surface opposite of the Ca 2+-and ligand-binding site [12][13][14][15]. This lack of microheterogeneity is unusual and may thus be structurally and/or functionally important.Enzymic removal of sialic acids provides a means to address the potential role(s) of this monosaccharide. With respect to catabolism, the desialylated form will be rapidly transported into hepacytes via asialoglycoprotein receptor-mediated endocytosi for lysosomal degradation [15]. Structurally, it retains its decameric state of aggregation and its full capacity to bind in vitro to amyloid fibrils and to DNA, whereas its interaction with agarose is reduced by 7% despite to opposite locations of the carbohydrate chain and the ligand-binding site [13,[15][16][17]. To answer the question, whether removal of the two sialic acid moieties from the single N-linked saccharide chain per promoter may cause a change in surface exposure of aromatic amino acids, a sensitive assay system is r...

Pentameric and decameric structures in solution of serum amyloid P component by X-ray and neutron scattering and molecular modelling analyses 1 1Edited by R. Huber

“…The uniform glycosylation of SAP may re¯ect the existence of speci®c carbohydrate interactions between the two pentamers. A laser-photoexcited dye study of SAP showed that the accessibility of some Trp, Tyr and His residues changes when SAP is desialylated (Siebert et al, 1995). Alterations in the glycosylation of different recombinant forms of SAP leads either to pentamers or decamers (unpublished results in Pepys et al, 1997), however the degree to which SAP glycosylation contributes to the formation of BAAB decamers is presently unclear.…”

NMR-Based, Molecular Dynamics- and Random Walk Molecular Mechanics-Supported Study of Conformational Aspects of a Carbohydrate Ligand (Galβ1-2Galβ1-R) for an Animal Galectin in the Free and in the Bound State

“…Even in the absence of a complete X-ray analysis for a lectin under investigation, docking studies can be performed with the help of available data on related agglutinins (44), as currently performed. Distinct predictions of such a modeling approach are being experimentally tested for the parameter of surface accessibility of tryptophan, tyrosine and histidine residues by the laser photo CIDNP (chemically induced dynamic nuclear polarization) technique (Siebert et al, in preparation), as recently applied to serum amyloid P component (45).…”

Knowledge-based Homology Modeling and Experimental Determination of Amino Acid Side Chain Accessibility by the Laser Photo CIDNP (Chemically Induced Dynamic Nuclear Polarization) Approach in Solution: Lessons from the Small Sialidase of Clostridium perfringens

“…CIDNP experiments were performed at 360 MHz on a Bruker AM-360 NMR spectrometer, as described in detail elsewhere [15][16][17]. CIDNP was induced by using flavin I mononucleotide as radical pair-generating dye.…”

“…Upon laser irradiation a CIDNP (chemically induced dynamic nuclear polarization) radical reaction takes place in the presence of e. g. a flavin derivative [14,15]. In glycosciences, this special technique has recently been instrumental to prove the occurrence of conformational changes after desialylation of human serum amyloid P component with only one invariant carbohydrate chain [16]. Moreover, the involvement of aromatic residues in the architecture of carbohydrate recognition domains in N-acetylglucosamine-binding lectins in solution has been documented with this approach [17].…”