The identification of proteinases of Porphyromonas gingivalis that act as virulence factors in periodontal disease has important implications in the study of host-pathogen interactions as well as in the discovery of potential therapeutic and immunoprophylactic agents. We have cloned and characterized a gene that encodes the 50-kDa cysteine proteinase gingipain or Arg-gingipain-1 (RGP-1) described previously (Chen, Z., Potempa, J., Polanowski, A., Wikstrom, M., and Travis, J. (1992) J. Biol. Chem. 267, 18896-18901). Analysis of the amino acid sequence of RGP-1 deduced from the cloned DNA sequence showed that the biosynthesis of this proteinase involves processing of a polyprotein that contains multiple adhesin molecules located at its carboxyl terminus. This finding corroborates previous evidence (Pike R., McGraw, W., Potempa, J., and Travis, J. (1994) J. Biol. Chem. 269, 406-411) that RGP-1 is closely associated with adhesin molecules, and that high molecular weight forms of the proteinase are involved in the binding of erythrocytes.

SUMMARYThe tumor suppressor maspin (mammary serpin) was originally identified as a component of human mammary epithelial cells that is downregulated as mammary tumor cells progress from the benign to the invasive and metastatic states. Maspin inhibits cellular invasion, motility, and proliferation, but its mechanism of action is currently unknown. Because the cellular machinery responsible for these processes is cytoplasmic, we have reexamined the tissue distribution and subcellular localization of maspin. We find that maspin, or a maspin-like protein, is present in many human organs, in which it localizes to epithelia. In cultured human mammary myoepithelial cells, maspin is predominantly a soluble cytoplasmic protein that associates with secretory vesicles and is present at the cell surface. In vitro assays show that the vesicle association is due to the existence of an uncleaved facultative secretion signal that allows small amounts of maspin to partition into the endoplasmic reticulum. These results demonstrate that maspin is more widespread than previously believed. The subcellular localization studies indicate that soluble intracellular and vesicleassociated maspin probably play an important role in controlling the invasion, motility, and proliferation of cells expressing it, whereas extracellular maspin may also regulate these processes in adjacent cells.

Enzyme replacement therapy (ERT) effectively reverses storage in several lysosomal storage diseases. However, improvement in brain is limited by the blood-brain barrier except in the newborn period. In this study, we asked whether this barrier could be overcome by higher doses of enzyme than are used in conventional trials. We measured the distribution of recombinant human ␤-glucuronidase (hGUS) and reduction in storage by weekly doses of 0.3-40 mg͞kg administered i.v. over 1-13 weeks to mucopolysaccharidosis type VII mice immunotolerant to recombinant hGUS. Mice given up to 5 mg͞kg enzyme weekly over 3 weeks had moderate reduction in meningeal storage but no change in neocortical neurons. Mice given 20 -40 mg͞kg three times over 1 week showed no reduction in storage in any area of the CNS except the meninges. In contrast, mice receiving 4 mg͞kg per week for 13 weeks showed clearance not only in meninges but also in parietal neocortical and hippocampal neurons and glia. Mice given 20 mg͞kg once weekly for 4 weeks also had decreased neuronal, glial, and meningeal storage and averaged 2.5% of wild-type hGUS activity in brain. These results indicate that therapeutic enzyme can be delivered across the blood-brain barrier in the adult mucopolysaccharidosis type VII mouse if administered at higher doses than are used in conventional ERT trials and if the larger dose of enzyme is administered over a sufficient period. These results may have important implications for ERT for lysosomal storage diseases with CNS involvement.␤-glucuronidase deficiency ͉ immune tolerance ͉ lysosomal storage disease ͉ mannose-6-phosphate receptor T he mucopolysaccharidoses (MPSs) are a group of lysosomal storage diseases (LSD) caused by the deficiency of enzymes needed for the stepwise degradation of glycosaminoglycans (GAGs). The widespread lysosomal accumulation of undegraded GAGs leads to progressive cellular and organ dysfunction (1). Current treatments for patients with MPSs include hematopoietic stem cell transplantation and enzyme replacement therapy (ERT) (2, 3). MPS type VII (also known as Sly disease) results from deficiency of ␤-D-glucuronoside glucuronosohydrolase (GUS; EC 3.2.1.31) and is inherited as an autosomal recessive trait. Affected patients share many clinical features with patients with other MPSs, including shortened life span, mental retardation, organomegaly, and bone and joint abnormalities, that are collectively referred to as dysostosis multiplex (4). The murine model of MPS VII has proven valuable for the evaluation of novel therapies for LSDs, including bone marrow transplantation, neural progenitor cell transplantation, somatic cell gene replacement therapy, and ERT (5).Previous studies have shown that i.v. injection of a fixed-dose of recombinant murine ␤-glucuronidase (mGUS) initiated at birth reduced pathological evidence of disease and prevented some of the learning, memory, and hearing deficits in the MPS VII mouse (6). However, recombinant mGUS reduced lysosomal storage in the neurons of the brain only i...