Worm Breeder's Gazette 13(2): 86 (February 1, 1994)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

bli-4 ,a unique member of the K ex2 -likeendoprotease family?

Martin Srayko}, Colin Thacker, Ann Rose

Figure 1

Department of Medical Genetics. University of British Columbia

Many biologically-active secreted proteins are synthesized as larger inactive precursors. Activation occurs during the secretion process by limited endoproteolytic cleavage, most frequently at pairs of basic amino acids such as Lys-Arg or Arg-Arg (reviewed by Docherty and Steiner, 1982. Annu. Rev. Physio. 44: 625-638). This excision is carried out by members of the K ex2 -likefamily of endoproteases. K ex2 pis a membrane-bound Ca(2+) dependent serine endoprotease identified in yeast.

Molecular characterization of bli-4 ,initiated by Ken Peters, indicates that this K ex2 -likegene has a unique feature among other known K ex2 -likemembers; bli-4 is the first example of a K ex2 -likegene in which two types of enzyme arise from the same gene via alternative splicing (WBG 11:28, 1991, and Thacker et al., this issue). Members of the K ex2 -likeendoprotease family can be divided into two classes based on the presence or absence of a transmembrane domain (TMD) near their carboxy terminus.

Blisterin A and B belong to the class of endoproteases that do not have a transmembrane domain, including PC2 , PC3 ,and PACE4 .The expression patterns for PC2 and PC3 have led to the suggestion that they may be involved in providing tissue specificity to prohormone processing.

Blisterin C falls into the class that contain a transmembrane domain, along with yeast K ex2 p,and the vertebrate and Drosophila furin proteins (Drosophila furin is also called dKLIP-1). In addition, other members of the family that contain a TMD are each found in a variety of tissue types. Because of their broad spectrum of expression, it has been suggested that both hfurin and PACE4 are components of the constitutive (tissue general) secretory pathway. In contrast, dKLIP-1 is predominantly expressed in the cortical regions of the adult central nervous system, although high levels of RNA are also detected in the ovary and developing embryo (Hayflick, et al., 1992. J. Neurosci. 12: 705-717).

Shown above are general structural features and relative lengths of the blisterins (as predicted by cDNA analysis), hfurin, PACE4 ,mouse PC3 ,human PC2 ,dKLIP-1 and yeast K ex2 ppolypeptides. Secretion signal peptides, SP, are shown as striped boxes; the protease domains are shown as dense dots with the positions of the catalytically important amino acids Asp, His, and Ser indicated by D, H, and S; the Cysrich regions are depicted as cross hatched boxes; transmembrane domains are shown as black boxes. Ovals indicate potential N-linked glycosylation sites. The potential autocatalytic cleavage sites are shown as vertical bars on the amino side of the protease domain. The numbers within the protease domains represent the percentage of identical residues when compared with the blisterins. Blisterin C cDNA did not contain a complete 5' end but is inferred to be identical to A and B in this region from Northern analysis.

The identification of K ex2 p-likeendoproteases in animal systems that are amenable to classical and molecular genetic manipulation provides an alternative approach for the examination of these important enzymes. In particular, we hope to utilize the large number of lethal mutations available for bli-4 to identify essential residues and/or important cis-acting regulatory elements. It is intriguing that bli-4 may provide both tissue general and tissue specific endoproteases from the same gene when one considers the variety of mutant phenotypes observed; bli-4 ( e937 )causes blistering in the adult cuticle, whereas all of the lethal alleles examined cause early larval or late embryonic arrest. We intend to further examine the expression of bli-4 isoforms via developmental Northern analysis and immunological localization studies.

Figure 1