Investigative Ophthalmology & Visual Science, Vol 36, 1306-1316, Copyright © 1995 by Association for Research in Vision and Ophthalmology

ARTICLES AND REPORTS

Identification and synthesis of chemotactic tripeptides from alkali- degraded whole cornea. A study of N-acetyl-proline-glycine-proline and N-methyl-proline-glycine-proline

RR Pfister, JL Haddox, CI Sommers and KW Lam
Eye Research Laboratories, Brookwood Medical Center, Birmingham, Alabama 35209, USA.

PURPOSE. To identify and synthesize the polymorphonuclear leukocyte chemoattractant(s) released from alkali-degraded corneas. METHODS. Corneas were degraded in 1.0 N NaOH, neutralized, ultrafiltered, and dialyzed. The final active ultrafiltrate was subjected to high- performance liquid chromatography on a Protein PAK I-60 column. The most active fractions were further separated on a mu-Bondapak-C18 and I- 60 column in sequence. RESULTS. Fraction 38 from the final I-60 column associated with a 210-nm absorption peak and elicited a polarization and chemotactic response from polymorphonuclear leukocytes. The loss of polarization activity in fraction 38 after exposure to prolidase suggests that this peptide contains a Pro-X (X = amino acid) peptide bond. The amino acid composition of fraction 38 was 35% glycine and 53% proline. Peptide sequence analysis was unable to establish a primary sequence even though Picotag analysis showed the presence of large amounts of the two amino acids. Mass spectrometry revealed only two molecular species of 312 MWt and 284 MWt. Tripeptides were synthesized using all possible amino acid permutations of 2 Pro and 1 Gly and tested in the polarization and chemotactic assays. These techniques demonstrated that n-acetyl-Pro-Gly-Pro, and to a lesser degree n-methyl- Pro-Gly-Pro, were the only synthetic tripeptides with activity similar to the purified chemoattractant. CONCLUSIONS. The data show that the chemotactic peptides, purified from alkali-degraded whole cornea and confirmed with identical synthetic tripeptides, are N-acetyl-Pro-Gly- Pro and N-methyl-Pro-Gly-Pro. Although a number of proteins contain the Pro-Gly-Pro sequence, large amounts of collagen in the cornea suggest this as a major source. The small size and hydrophilic nature of these chemoattractants are predictive of a high degree of diffusibility. These chemoattractants are likely to play a major role in the early neutrophil response after an alkali injury.

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