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1. A series of eight apoSAA1.1 peptides corresponding to wild-type mouse apoSAA1.1, residues 77-103, (m27mer) six peptides in which one or more of the basic residues were replaced with A, and one 20mer residues 77-96 (missing K102) were synthesized by Multiple Peptide Systems (San Diego, California, U.S.A.). (see Notes 1 and 2).

Fig. 3. Determination of the GAG-binding specificity for m27mer. Heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), and hyaluronan (HA) were covalently linked to either Sepharose 4B of Affigel 102. The elution profiles on DS-Sepharose and HA-Sepharose (asterisks) were very similar to that of CS-Sepharose.

Fig. 3. Determination of the GAG-binding specificity for m27mer. Heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), and hyaluronan (HA) were covalently linked to either Sepharose 4B of Affigel 102. The elution profiles on DS-Sepharose and HA-Sepharose (asterisks) were very similar to that of CS-Sepharose.

m27mer (mouse apoSAA1.177-103)







K102- (mouse apoSAA1.177-96)


2. Purity of the peptides was analyzed by RP-HPLC and, where required, the peptide was re-purified.

4. Notes

To localize GAG-binding sites to specific peptide sequences, limited chemical or enzymatic cleavage is required of the native protein. Additional flanking sequences to maintain appropriate secondary structure and cleavage near basic residues (K, H, R) should be avoided. Chemical cleavage at M-X with CNBr, or at W-X with BNPA-skatole is a good starting point, since both residues occur with relatively low frequency in proteins (M 2.4% and W 1.3%) (16).

2. Whenever possible, conservative residue replacements should be performed. The strategy for deciding on replacement of residues in GAG-binding sites should take into account potential changes in peptide solubility, secondary structure (i.e., conformational preferences a-helix, p-strand, p-bend turn), and the volume of the amino acid chosen. The basic residues most often shown to be crucial for GAG binding (K, H, R) are a-helix formers (16), and A can be a satisfactory replacement. However, if solubility of the peptide is adversely affected then Q may be a useful alternative. Also, Qs van der Waals volume is more similar to K, H, and R than A; Q = 114 A3 vs A = 69 A3, compared to K = 135 A3, H = 118 A3, and R = 148 A3.

3. All GAGs, CCS, DS, HA, HS, and heparin can be covalently coupled to column matrices, and determination of the GAG-binding specificity of any peptide using these columns can be easily carried out. Heparin is quite popular and inexpensive, and is often used as a cheap substitute for HS. While heparin and HS are synthesized in a similar fashion, sulfation and uronic acid epimer (glucuronic vs iduronic acid) content and distribution are quite different. Also HS is most often the physiological ligand for most proteins and peptides that can bind heparin. We have observed that while heparin-binding activity was useful in localizing the GAG-binding site on apoSAA1.1, the minimal peptide sequence required for binding to heparin was at least 7 residues shorter then that required for HS binding. In addition, deletion of K102 destroyed binding for HS-Sepharose, but not for heparin-Sepharose implying that the K102-peptide was binding to different oli-gosaccharide sequences.

4. Type of support matrix. Sepharose vs Affigel helps define the type of chemical linkage group available to react with one or more of the GAG's reactive side groups. This is an important factor, since the linkage of GAG to matrix can influence binding activity. We found that the basic residue requirements were found to be different for heparin, depending on whether it was linked via its COO- or HO- groups. We observed that K102-bound heparin-Sepharose but not heparin-Affigel. A similar discrepancy was seen with the m27mer peptide, which bound HS only when linked to Sepharose, not Affigel. Furthermore, treatment of HS-Sepharose with carbodiimide indicated that blocking of the car-boxyls on HS was likely responsible for the loss of binding activity.

5. Preliminary screening of protein/peptide for GAG-binding activity can be carried out relatively quickly with 1- to 10-mL columns. Samples can be loaded in 0-0.15 M NaCl, followed by an increasing linear NaCl concentration gradient to 1 or 2 M NaCl totaling 10-20 bed volumes to develop the elution profile. Distinguishing between different pep-tides with similar affinities for the GAG column was more readily achieved with the columns connected to HPLC. The HPLC allowed for more precise solvent delivery and gradient formation, highly reproducible retention times, and accurate determination of NaCl concentrations required for protein/peptide desorption.

6. FPLC- and HPLC-grade heparin columns are also available from a number of suppliers.


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