On the structural significance of the linkage region constituents of N-glycoproteins: an X-ray crystallographic investigation using models and analogs

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Abstract

The linkage region constituents, namely, 2-acetamido-2-deoxy-β-d-glucopyranose and asparagine are conserved in the N-glycoproteins of all the eukaryotes. The present work is aimed at understanding the reasons for the occurrence of GlcNAc and Asn as the linkage region constituents. A total of six sugar amides have been designed as models and analogs of the linkage region and their crystal structures have been solved. This is the first report on the X-ray crystallographic investigation of the effect of systematic changes in the linkage sugar as well as its aglycon moiety on the N-glycosidic torsion, ψN (O5–C1–N1–C1). This also forms the first report on the crystal structure of a model of l-RhaβAsn, a variant linkage found in the surface layer glycoprotein of Bacillus stearothermophillus. Among the models and analogs examined, the acetamido derivatives of Man and Xyl, the linkage sugars of O-glycoproteins, show a ψN value of −114.5° and −121.2°, respectively, deviating maximum from the value of −89.8° reported for the model compound GlcNAcβNHAc. The l-Rha and Gal derivatives also show noticeable deviations. The ψN values, −89.5° and −91.0°, of the propionamide derivatives of Glc and GlcNAc (analogs of GlcβGln and GlcNAcβGln, respectively) agree well with those (−93.8° and −89.8°) reported for their corresponding acetamide derivatives suggesting Gln could serve as well as Asn as the linkage region amino acid. However, the rotational freedom about the additional C–C bond would lead to altered rigidity of the linkage region. An analysis of packing reveals that the molecular assembly of these compounds is driven by different infinite and finite chains of hydrogen bonds. The double pillaring of hydrogen bonds involving the amide groups at C1 and C2 is seen as a unique packing feature characteristic of β-1-N-acyl derivatives of GlcNAc. Based on the findings of the present study, it is speculated that the linkage region constituents of the eukaryotic N-glycoproteins appear to fulfill three essential structural requirements: rigidity, planarity, and linearity and these are met by the trisaccharide core and Asn at the linkage region.

Section snippets

Results and discussion

Among the various sugar amides (Fig. 2) designed in the present work, GlcβNHPr (1) and GlcNAcβNHPr (2) are analogs of the respective sugar conjugates of glutamine. l-RhaβNHAc (3) is the model of l-RhaβAsn, the unusual N-glycosidic linkage found in the crystalline surface layer glycoproteins of the eubacterium B. stearothermophillus[6d]. Man, Gal, and Xyl are the common linkage region sugars attached to Ser in O-glycoproteins. In an effort to evaluate the conformational preferences of these

Linkage region conformation

The value, 172.3°(3), of χ1 (N1–C1–C2–C3) for the propionamide derivative (1) reveals the anti conformation about C1–C2 bond and compares well with that of 166.5° reported for GlcβAsn · H2O [12b]. The corresponding value observed for 2 turns out to be 114.7°(7) differing by about 73.2° from the value of 187.9° observed for GlcNAcβAsn · 3H2O. This large difference in the χ1value is noteworthy. The Z conformation about the amide linkage (N1–C1) in all the compounds is evident from the torsion ψN

Molecular packing and hydrogen bonding

Hydrogen bonding plays a major role in the structural and hence the functional attributes of all the three major classes of biomolecules—proteins, nucleic acids, and carbohydrates. Not surprisingly, these classes of compounds are found in aqueous environments resulting in further modifications owing to the formation of an intense network of intermolecular hydrogen bonding. In proteins, the number of intramolecular hydrogen bonding components namely the amide groups is about one for every four

Conclusions

Glycosylation occurs without exception in the entire phylogenetic spectrum, ranging from archaea and eubacteria to eukaryotes. Formation of the sugar–amino acid linkage is a vital event in the biosynthesis of glycoproteins. The linkage region, GlcNAcβAsn, is conserved in all the eukaryotic N-glycoproteins. The present work is aimed at understanding the structural significance of GlcNAc and Asn as the linkage region constituents. The study is focused on the glycosidic torsion, ψN (O5–C1–N1–C1),

Experimental

All the sugars used in the study are d-sugars unless mentioned otherwise. Synthesis of the sugar amides 1 and 2 was accomplished from the glycosylamines by selective N-acylation [21a] while the remaining amides were prepared by peracylation followed by de-O-acylation [21b]. Crystallization was done in aqueous methanol at room temperature. X-ray diffraction data were collected at room temperature in the ω−2θ scan mode on an Enfra-Nonius CAD4 diffractometer and the relevant details of data

Acknowledgements

This work was supported by the Department of Science and Technology, New Delhi. T.L. thanks the CSIR, New Delhi, for the Junior and Senior Research Fellowships during the course of this work. We acknowledge Dr. Babu Varghese for his valuable technical help and Mr. M.V. Chandrasekar for his assistance in preparing a few of the compounds. We thank Professor S. Srinivasan, Professor V.S.R. Rao, and Professor P.K. Qasba for their valuable suggestions and the RSIC, IIT Madras for data collection.

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