Yolk hydrolase activities associated with polypeptide and oligosaccharide processing of Blattella germanica vitellin

Abstract

Various aspects of the processing of Blattella germanica vitellin (Vt) in the oocyte and egg have been investigated. Employing subunit specific antibodies, the precursor product relationships among t h e subunits of this Vt have been determined. After endocytosis of Vt by the oocyte, t h e M, 160,000 subunit Vt is cleaved to products of M, 95,000 and M, 50,000. In association with an unprocessed M, 102,000 peptide, these form the subunits of the Vt of freshly ovulated eggs. Between 4 and 5 days post ovulation (at 3OoC), all three subunits of Vt are again processed proteolytically before use by the embryo. Although Vt's high mannose-type oligosaccharides are trimmed during embryogenesis, their modification occurs subsequent to the day 4-5 proteolysis, precluding the possibility that changes in oligosaccharide content or structure contribute to regulating this second proteolytic event.Although the predominant oligosaccharide of Vt is Man9GlcNAc2, the M, 50,000 subunit of egg-borne Vt contains a much higher proportion of Man6GlcNAc2 than t h e other two subunits; therefore, this portion of the precursor vitellogenin must be more accessible to the processing mannosidases of the endoplasmic reticulum during its biosynthesis. A microtechnique for aspirating the yolk from individual eggs in an ootheca permits its isolation free of contamination by embryonic tissue. With this procedure, the specific activity profiles of exo-a-rnannosidase, exo-0-Nacetylgl ucosam i n idase, a-glucosidase and acid phosphatase were monitored during the first 6 days after ovulation, and some of their properties were also determined. Expression of the acid phosphatase and exo-P-N-acetyl- glucosarninidase activities coincide with the day 4-5 proteolysis, while arnannosidase remains relatively constant throughout the first 6 days.Functions for these enzymes and the oligosaccharides of Vt during Vt storage and utilization are proposed.

A Cysteine Protease That Processes Insect Vitellin

“…Unique limit peptides in vitro were M r 88,000, 70,000, and 50,000 -53,000. Processing of the M r 50,000 subunit in vitro was obscured by other products of that approximate size, but this subunit is processed beginning at day 5 (16,18). Addition of fresh enzyme to the in vitro incubation did not alter the product distribution.…”

“…Previous work demonstrated that Vt utilization in Blattella germanica eggs is initiated at days 3-4 postovulation by proteolytic processing to a distinct set of peptides, which are then accessed by the embryo during development (9,15,16). Similar processing events are characteristic of Vt utilization in other insect species also (see Ref.…”

“…11, lanes C, minus endo H). Removal of carbohydrate by endo H, indicated by a slight increase in mobility (lanes C, plus and minus endo H) suggests that most, if not all, occurs as high Man-type oligosaccharides (30), the class of oligosaccharides found in B. germanica Vt (16,35) and other insect glycoproteins (36,37). The probe also labeled the endo H-treated protein, but with less intensity than with the control, a result reflecting either incomplete removal of oligosaccharides or to the remaining single GlcNAc residues (30).…”

“…The M r 29,000 band's mobility matched that of the purified protease (lane 2). Day 0 yolk (lane 3) was labeled more extensively by the probe, especially the three Vt subunits at M r 102,000, 95,000, and 50,000, which have been degraded by day 6 in vivo (16). However, lane 3 also contains three probe-reactive peptides at approximately M r 40,000 (arrowheads), absent when day 0 yolk was preincubated with E-64 (lane 4).…”

“…Electrophoresis-SDS-PAGE was conducted by a modification (25) of the Laemmli method (26) at 75 mA/gel as described previously (16). Nondenaturing PAGE was run at 200 V by a modification (Sigma bulletin no.…”