Fig 2: CRISP domain organisation and tertiary structure.(A) CRISP domain organisation and (B) homology models of mouse and human CRISP3 based on the crystal structure of Natrin. The CRISP domain is shown in blue/aqua; disulfide bonds by white/yellow linkages. N-glycosylation is represented by high mannose (mouse CRISP3) and complex (human CRISP3) glycans (ball and stick models).

Fig 3: Mouse CRISP3 is incompletely digested by Endo H.(A) Mouse CRISP3 wild type, N99Q and N113Q are partially digested by Endo H (E). Complete digestion with PNGase F (P) is included for size comparison. (B) A size comparison of mouse and human CRISP3 (mCRISP3 and hCRISP3, respectively) glycans (square brackets). Full length gels are available under supplementary data; gels were run under the same conditions.

Fig 4: CRISP3 from whole mouse salivary gland is unglycosylated.Western blot comparison of wild type salivary gland CRISP3 with recombinant mouse CRISP3 glycosylated at two sites (wild type, lane 1), one site (N99Q, lane 2) and unglycosylated (N99Q + N113Q + N156Q, lane 3). Full length gel is available under supplementary data.

Fig 5: Recombinant mouse CRISP3 is glycosylated at N99 and N113.Western blotting with His tag antibody showed glycosylated human and mouse CRISP3-His to be 29 and 33 kDa respectively (A). (B) Peptide pre-absorption of mouse CRISP3 antibody. (C) Mouse CRISP3 N99Q and N113Q mutant proteins were smaller than wild type (WT) mouse CRISP3 (PNGase F untreated samples); N156Q was unaffected. Two mutants unable to undergo glycosylation (N99Q + N113Q and N99Q + N113Q + N156Q) were of relatively low abundance. ‘NT' represents non-transfected HEK cell media. Full length gels are available under supplementary data; gels were run under the same conditions.