Johannes A. Kramps

The polypeptide chains of α-crystallin from old human eye lenses☆

Abstract The polypeptide chains of α-crystallin from 60- to 80-year-old human eye lenses were studied to determine whether they are subject to similar post-synthetic alterations as known from bovine α-crystallin. Isoelectric focusing in the presence of 6 M urea revealed that old human α-crystallin consists of five major polypeptide chains, designated as Ax, A2, Bx, By and B2. By dodecyl sulphate gel electrophoresis mainly chains with apparent molecular weights of 22 000 and 20 000 were observed. Ion-exchange chromatography in the presence of urea was applied to obtain enriched preparations of individual polypeptides. Additional data concerning the primary structure of some chains were gathered by tryptic peptide mapping in combination with amino acid analysis. The presence of two cysteine residues in the A-chains was demonstrated. The Ax component turned out to be composed of two chains: deamidated A2 of normal length (172 residues) and, presumably, a shortened A2-chain: A21–151. The Bx- and By-chain with isoelectric points slightly lower and higher, respectively, than that of bovine B1, appeared to be B21–170 and deamidated B2 of normal length (175 residues). A group of polypeptides with very acidic isoelectric points, present in low proportion, showed a heterogeneous molecular weight distribution below 18 000 and a high degree of similarity with the A-chains as judged by tryptic peptide mapping. In spite of several differences in subunit composition, it can be concluded that in human α-crystallin, as in bovine α-crystallin, deamidation and degradation processes occur.

Proteinase inhibitory activities of antileukoprotease are represented by its second COOH-terminal domain

Antileukoprotease or secretory leukocyte proteinase inhibitor is a potent serine proteinase inhibitor produced by exocrine glands of the human body. This monomeric protein (107 amino acids) comprises two homologous domains. It is generally thought that Leu19-Arg20-Tyr21 in the NH2-terminal domain represent the trypsin inhibitory activity, whereas Leu72-Met73-Leu74 in the COOH-domain represent the chymotrypsin and elastase inhibitory activity. Besides Met73, antileukoprotease contains three additional methionine residues all located in the COOH-terminal domain. Treatment of antileukoprotease with different amounts of methionine-selective reagents such as myeloperoxidase in the presence of H2O2 and Cl-, or cis-platinumdiammine dichloride resulted in a dose-dependent inactivation of all inhibitory activities, suggesting that methionine residues are involved in these activities. By using specific synthetic substrates, it was observed that elastase is able to displace trypsin from the inhibitor molecule, indicating that the trypsin and elastase inhibitory sites are located close to each other or at the same site. Incubation of antileukoprotease or its recombinant COOH-terminal domain with an antileukoprotease-specific monoclonal antibody (MoAb15) resulted in a strong selective increase of the trypsin inhibitory activity. The results presented reveal strong evidence that the inhibitory activities of antileukoprotease against trypsin, chymotrypsin and elastase are represented by its COOH-terminal domain, and that methionine residues are involved in interactions with these proteinases.

The primary structure of the B2 chain of human α‐crystallin

a-Crystallin is one of the major proteins of the mammalian eye lens (for review see [ 11). The composing polypeptide chains, A and B, of this structural protein undergo extensive modifications upon ageing, both in the ox [2,3] and, much more pronounced, in man [4,5]. To understand these processes, which may also be relevant to the formation of cataract, it is necessary to know the structures of the A and B chains. The primary structure of human cy-crystallin A chain has previously been determined [6]. This letter shows that the human Bz chain differs only in three positions from the already known bovine B2 chain [7], thus demonstrating that the B chain, like the homologous A chain, has a very slow rate of evolutionary change. The knowledge of the primary structure of human Bz chain will facilitate the characterization of the B-like chains which appear upon ageing in the human lens.

Intratracheally-instilled antileukoprotease and α1-proteinase inhibitor: effect on human neutrophil elastase-induced experimental emphysema and pulmonary localization

SummaryThe protective capacities of intratracheally-instilled antileukoprotease and α1-proteinase inhibitor towards human neutrophil elastase (HNE)-induced pulmonary injuries were compared in hamsters. The antiproteases were instilled in equimolar amounts up to 20 h before HNE instillation. At all intervals, both inhibitors were able to inhibit HNE-induced emphysema efficiently. At l h before HNE instillation, α1-proteinase inhibitor was more effective in this regard than antileukoprotease.α1 -Proteinase inhibitor, instilled 1 to 12 h before HNE, efficiently inhibited HNE-induced haemorrhage, while the antileukoprotease protected haemorrhage only when it was administered 1 h before HNE. The development of secretory cell metaplasia was affected only when both inhibitors were instilled 1 h before HNE. In a second series of experiments, the localization of the two antiproteases after intratracheal instillation in hamster was investigated using an indirect immunofluorescence technique. Up to 20 h after installation, antileukoprotease was found to be associated with elastin fibres at all points of time investigated. In contrast, α1-proteinase inhibitor was observed to be located in the alveolar lining and diffusely in the alveolar lung tissue at all points of time investigated. No association of the inhibitor with elastin fibres was found. We conclude that the fraction of antileukoprotease associated with the elastic fibre may be important in the protection of HNE-induced pulmonary emphysema.