Characterizing the N-Glycosylation of Corticotropin Releasing Factor-Binding Protein

A. G. Craig, S. W. Sutton, J. Vaughan, S.C. Koerber and W. H. Fischer.

The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, P.O. Box 85800, San Diego, CA 92186-5800.

Corticotropin Releasing Factor-Binding Protein (CRF-BP) is a 35 kDa protein originally purified from human plasma which has been proposed to protect the maternal endocrine system from high levels of CRF produced by the placenta during pregnancy. CRF-BP inhibits CRF-induced ACTH release from pituitary cells in vitro (1) . Based on the protein sequence, Asn180 was proposed as a site of N-linked glycosylation.

We have investigated the use of anti CRF-BP serum and matrix assisted laser desorption (MALD) to measure CRF-BP recombinantly expressed in Chinese Hamster Ovary Cells. We observed inhibition of ion formation in MALD spectra measured from rCRF-BP in a variety of forms (affinity and reverse phase purified) In contrast, immunoprecipitation of rCRF-BP (incubation overnight at 4°C in a 50 mM Hepes buffer with antisera raised against either an N-terminal CRF-BP peptide or the intact rCRF-BP) and rinsing of the pellet formed (20 min. centrifuge @ 15000 rpm) with 50 mM Hepes buffer (x2) and subsequent addition of 5 ml of 0.1 % aqTFA and application of an aliquot (0.5 ml) of this solution to a thin film of a-cyano-4-hydroxy cinnamic acid resulted in intact CRF-BP ion formation (measured with a Bruker Reflex operated in the linear mode) [Intact ions observed at m/z 8906, 11758, 17632 and 35445. It is possible that the elimination of MALD inhibition is due to the the specificity of the immunoprecipitation resulting in a rinsing from the sample of contaminants or salts which are not removed by other purification procedures. Alternatively, the antibody precipitation may be altering a more intrinsic property of the protein relating to its amenability for ionization. Clearly, the presence of the antibody does not inhibit ion formation. Using this sample preparation technique we have measured the intact mass of recombinant CRF-BP after enzymatic treatment with peptide N-glycosidase-F (PNGase-F) [Intact ions observed at m/z 8417, 11213, 16865 and 33872], indicating that the glycan molecule mass is approximately 1.5-2 kDa.

In addition, the CRF-BP glycopeptide has been identified from a tryptic map of CRF-BP in order to obtain more detailed information on the nature of the glycan structure and to verify that Asn180 is the only site of glycosylation. The MALD mass spectrum of the 180-192 fragment which was obtained after tryptic digestion followed by TCEP reduction (2) contained 2 species at m/z 3402 and 3691 , while the spectrum of the same fraction isolated after PNGase-F treatment contained a single species at m/z 1484.1. The mass shift observed indicates a more accurate mass of 2207 Da for the major glycan species, while the intense fragment ion (m/z 3402) is characteristic for the presence of sialic acid (3) ,. Using the previously proposed strategy of Huberty et al (3) , we searched for N-linked glycan structures containing sialic acid in The Complex Carbohydrate Structure Database (4, 5) and found that only two previously reported structures are consistent with the observed glycan mass. These two structures, (Gal)2(Man)3(GlcNAc)4(Neu)2 (2206 Da) and (Fuc)2(Gal)2(Man)3(GlcNAc)4(Neu) (2207 Da), differ in the degree of sialic acid present (one or two sialic acid molecules). After treatment of the 180-192 glycopeptide with neuraminidase the MALD mass spectrum contained a species consistent with loss of two sialic molecules from the glycan (data not shown). On the basis of these measurements we propose that the glycosylation present on CRF-BP is consistent with the previously reported glycan structure (Gal)2(Man)3(GlcNAc)4(Neu)2.

References
1. Potter, E.; Behan, D. P.; Fischer, W. H.; Linton, E. A.; Lowry, P. J.and Vale, W. W. (1991) Nature 349, 423-426.
2. Fischer, W. H.; Rivier, J. E.and Craig, A. G. (1993) Rapid Commun Mass Spectrom 7, 225-228.
3. Huberty, M. C.; Vath, J. E.; Yu, W.and Martin, S. A. (1993) Anal Chem 65, 2791-2800.
4. Doubet, S.and Albersheim, P. (1992) Glycobiology 2, 505-507.
5. Van Kuik, J. A.and Vliegenthart, J. F. G. (1992) Trends Biotechnol 10, 182-185.