Targeting Iron - Respiratory Reciprocity Promotes Bacterial Death.

Discovering new bacterial signaling pathways offers unique antibiotic strategies. Here, through an unbiased resistance screen of 3,884 gene knockout strains, we uncovered a previously unknown non-lytic bactericidal mechanism that sequentially couples three transporters and downstream transcription to lethally suppress respiration of the highly virulent P. aeruginosa strain PA14 - one of three species on the WHO's 'Priority 1: Critical' list. By targeting outer membrane YaiW, cationic lacritin peptide 'N-104' translocates into the periplasm where it ligates outer loops 4 and 2 of the inner membrane transporters FeoB and PotH, respectively, to suppress both ferrous iron and polyamine uptake. This broadly shuts down transcription of many biofilm-associated genes, including ferrous iron-dependent TauD and ExbB1. The mechanism is innate to the surface of the eye and is enhanced by synergistic coupling with thrombin peptide GKY20. This is the first example of an inhibitor of multiple bacterial transporters.

AUTOPHAGY IN THE EYE: FROM PHYSIOLOGY TO PATHOPHYSOLOGY.

Autophagy is a catabolic self-degradative pathway that promotes the degradation and recycling of intracellular material through the lysosomal compartment. Although first believed to function in conditions of nutritional stress, autophagy is emerging as a critical cellular pathway, involved in a variety of physiological and pathophysiological processes. Autophagy dysregulation is associated with an increasing number of diseases, including ocular diseases. On one hand, mutations in autophagy-related genes have been linked to cataracts, glaucoma, and corneal dystrophy; on the other hand, alterations in autophagy and lysosomal pathways are a common finding in essentially all diseases of the eye. Moreover, LC3-associated phagocytosis, a form of non-canonical autophagy, is critical in promoting visual cycle function. This review collects the latest understanding of autophagy in the context of the eye. We will review and discuss the respective roles of autophagy in the physiology and/or pathophysiology of each of the ocular tissues, its diurnal/circadian variation, as well as its involvement in diseases of the eye.

Lacripep for the Treatment of Primary Sjögren-Associated Ocular Surface Disease: Results of the First-In-Human Study.

PURPOSE: The purpose of this study was to assess the safety, tolerability, dosing, and efficacy of the active 19 amino acid fragment of lacritin (Lacripep), a broad regulator of ocular surface homeostasis, in the treatment of ocular surface disease associated with primary Sjögren syndrome. METHODS: Two hundred four subjects were randomized to receive vehicle, 22 µM Lacripep, or 44 µM Lacripep 3 times daily for 28 days, preceded by a 14-day run-in and followed by 14-day washout. Outcome measures were corneal fluorescein staining (CFS), lissamine conjunctival staining, Schirmer with anesthesia, tear break-up time, SANDE scoring, and visual analog scale assessment of symptoms. RESULTS: This study established the safety and tolerability of topical treatment with Lacripep in patients with primary Sjögren syndrome. There were few adverse events: Only mild irritation was found in less than 3 percent of patients dosed with Lacripep. Total CFS and Eye Dryness Score were not significantly changed at day 28. Post hoc analysis of patients with Eye Dryness Severity scores of 60 or greater at baseline revealed significant improvements in inferior CFS at 14 and 28 days and complaints of burning and stinging at 14 days. Significant improvement in regional lissamine conjunctival staining was seen at 14 and 28 days. CONCLUSIONS: This first-in-human study of Lacripep in patients with primary Sjögren syndrome demonstrated clinically significant improvements in specific signs and symptoms on which to base future studies. This study established safety and tolerability and potential metrics of efficacy in patients with moderate to severe disease. Further work on appropriate dosing and concentration is ongoing.

Autophagy in the normal and diseased cornea.

The cornea and covering tear film are together the 'objective lens' of the eye through which 80% of light is refracted. Despite exposure to a physically harsh and at times infectious or toxic environment, transparency essential for sight is in most cases maintained. Such resiliency makes the avascular cornea a superb model for the exploration of autophagy in the regulation of homeostasis with relevancy to all organs. Nonetheless, missense mutations and inflammation respectively clog or apparently overwhelm autophagic flux to create dystrophies much like in neurodegenerative diseases or further exacerbate inflammation. Here there is opportunity to generate novel topical therapies towards the restoration of homeostasis with potential broad application.

Lacritin proteoforms prevent tear film collapse and maintain epithelial homeostasis.

Lipids in complex, protein-enriched films at air/liquid interfaces reduce surface tension. In the absence of this benefit, the light refracting and immunoprotective tear film on eyes would collapse. Premature collapse, coupled with chronic inflammation compromising visual acuity, is a hallmark of dry eye disease affecting 7 to 10% of individuals worldwide. Although collapse seems independent of mutation (unlike newborn lung alveoli), selective proteome and possible lipidome changes have been noted. These include elevated tissue transglutaminase and consequent inactivation through C-terminal cross-linking of the tear mitogen lacritin, leading to significant loss of lacritin monomer. Lacritin monomer restores homeostasis via autophagy and mitochondrial fusion and promotes basal tearing. Here, we discover that lacritin monomer C-terminal processing, inclusive of cysteine, serine, and metalloproteinase activity, generates cationic amphipathic α-helical proteoforms. Such proteoforms (using synthetic peptide surrogates) act like alveolar surfactant proteins to rapidly bind and stabilize the tear lipid layer. Immunodepletion of C- but not N-terminal proteoforms nor intact lacritin, from normal human tears promotes loss of stability akin to human dry eye tears. Stability of these and dry eye tears is rescuable with C- but not N-terminal proteoforms. Repeated topical application in rabbits reveals a proteoform turnover time of 7 to 33 h with gradual loss from human tear lipid that retains bioactivity without further processing. Thus, the processed C-terminus of lacritin that is deficient or absent in dry eye tears appears to play a key role in preventing tear film collapse and as a natural slow release mechanism that restores epithelial homeostasis.

Development of a Quantitative Immunoassay for Tear Lacritin Proteoforms.

Purpose: Lacritin is a tear glycoprotein with pro-tearing and pro-ocular surface homeostasis activities that is selectively deficient in most dry eye tears. Proteoforms include an active monomer, inactive polymers, and a splice variant termed lacritin-c. Quantitation of the different proteoforms of tear lacritin may provide a diagnostic tool for ocular diseases. Here, we report the development of an immunoassay for the quantification of multiple lacritin proteoforms in human tear samples. Methods: Basal tears collected on Schirmer test strips with anesthesia were eluted by diffusion and centrifugation under optimized conditions. Tear protein concentrations were determined, and 2.56 µg of each sample was separated by SDS-PAGE followed by western blot analysis. Blots were challenged with anti-Pep Lac N-term antibodies. Detection was with fluorescent secondary antibodies visualized by the LI-COR Odyssey CLx imaging system and quantified with standard curves of recombinant lacritin. Results: The percent total lacritin (ng lacritin/100 ng total protein) ranged from 1.8% to 14.8%. Monomer, lacritin-c, and polymer proteoform percent total protein ranged from 1.1% to 6.3%, 0.3% to 5.4%, and 0.7% to 5.7%, respectively. Monomer lacritin was detected at concentrations of 6 to 176 µM, with lacritin-c and polymer proteoforms at 2 to 46 µM and 1 to 23 µM, respectively. Conclusions: This assay greatly exceeds the power and sensitivity of our prior lacritin enzyme-linked immunosorbent assay that was not capable of distinguishing monomer from polymers and lacritin-c proteoforms. Translational Relevance: A new method has been developed to quantitate multiple proteoforms of tear lacritin in preparation for analyses of samples from clinical trials.

Biosynthesized Multivalent Lacritin Peptides Stimulate Exosome Production in Human Corneal Epithelium.

Lacripep is a therapeutic peptide derived from the human tear protein, Lacritin. Lacripep interacts with syndecan-1 and induces mitogenesis upon the removal of heparan sulfates (HS) that are attached at the extracellular domain of syndecan-1. The presence of HS is a prerequisite for the syndecan-1 clustering that stimulates exosome biogenesis and release. Therefore, syndecan-1-mediated mitogenesis versus HS-mediated exosome biogenesis are assumed to be mutually exclusive. This study introduces a biosynthesized fusion between Lacripep and an elastin-like polypeptide named LP-A96, and evaluates its activity on cell motility enhancement versus exosome biogenesis. LP-A96 activates both downstream pathways in a dose-dependent manner. HCE-T cells at high confluence treated with 1 µM LP-A96 enhanced cell motility equipotent to Lacripep. However, cells at low density treated with 1 µM LP-A96 generated a 210-fold higher number of exosomes compared to those treated at low density with Lacripep. As monovalent Lacripep is capable of enhancing cell motility but not exosome biogenesis, activation of exosome biogenesis by LP-A96 not only suggests its utility as a novel molecular tool to study the Lacritin biology in the corneal epithelium but also implies activity as a potential therapeutic peptide that can further improve ocular surface health through the induction of exosomes.

The Lacritin-Syndecan-1-Heparanase Axis in Dry Eye Disease.

Homeostasis and visual acuity of the surface of the eye are dependent on tears, a thin film comprising at least 1800 different extracellular proteins and numerous species of lipids through which 80% of entering light is refracted at the air interface. Loss of homeostasis in dry eye disease affects 5-7% of the world's population, yet little is known about key molecular players. Our story began as an unbiased screen for regulators of tearing that led to the discovery of homeostasis-restorative 'lacritin', a tear protein whose active form is selectively deficient in dry eye. Heparanase acts as a novel 'on-switch' for lacritin ligation of syndecan-1 necessary to trigger basal tearing, as well as pertussis toxin-sensitive and FOXO-dependent signaling pathways for healing of inflammation-damaged epithelia and restoring epithelial oxidative phosphorylation by mitochondrial fusion downstream of transiently accelerated autophagy. A phase 2 clinical trial has tested the applicability of this mechanism to the resolution of dry eye disease. Results are not yet available. With lacritin proteoforms detected in cerebral spinal fluid, plasma, and urine, the capacity of the lacritin-syndecan-1-heparanase axis to restore homeostasis might have wide systemic relevance to other organs.

Thermally-Responsive Loading and Release of Elastin-Like Polypeptides from Contact Lenses.

Contact lenses are widely prescribed for vision correction, and as such they are an attractive platform for drug delivery to the anterior segment of the eye. This manuscript explores a novel strategy to drive the reversible adsorption of peptide-based therapeutics using commercially available contact lenses. To accomplish this, thermo-sensitive elastin-like polypeptides (ELPs) alone or tagged with a candidate ocular therapeutic were characterized. For the first time, this manuscript demonstrates that Proclear CompatiblesTM contact lenses are a suitable platform for ELP adsorption. Two rhodamine-labelled ELPs, V96 (thermo-sensitive) and S96 (thermo-insensitive), were employed to test temperature-dependent association to the contact lenses. During long-term release into solution, ELP coacervation significantly modulated the release profile whereby more than 80% of loaded V96 retained with a terminal half-life of ~4 months, which was only 1-4 days under solubilizing conditions. A selected ocular therapeutic candidate lacritin-V96 fusion (LV96), either free or lens-bound LV96, was successfully transferred to HCE-T cells. These data suggest that ELPs may be useful to control loading or release from certain formulations of contact lenses and present a potential for this platform to deliver a biologically active peptide to the ocular surface via contact lenses.

Development of lacrimal gland inflammation in the mouse model of herpes stromal keratitis.

Herpes stromal keratitis (HSK) is a chronic immunoinflammatory condition which develops in response to recurrent herpes simplex virus-1 (HSV-1) infection of the cornea. Patients with HSK often demonstrate the concurrence of corneal desiccation and the loss of blink reflex. However, the relationship between severity of HSK, level of basal tears and inflammation of the lacrimal gland is mostly unexplored. In this study, we compared these variables in extraorbital lacrimal gland (EoLG) after corneal HSV-1 infection in the C57BL/6J mouse model. Our results showed a significant reduction in the volume of tears in infected eyes during the development of HSK. Extensive architectural damage to EoLG, presumably caused by a massive influx of interferon-gamma secreting T cells, was observed during clinical disease period of HSK. A positive correlation between the decrease in tear volume, severity of HSK and the damage to EoLG were evident in infected mice. The presence of infectious virus measured in EoLG during pre-clinical, but not clinical disease period of HSK, suggested that viral cytopathic effects are not the major contributors of extensive damage seen in EoLG. Furthermore, topical administration of lacritin peptide delayed but did not prevent the decrease in tears in HSV-1 infected mice, and had no significant effect in either reducing the severity of HSK or T cell infiltration in EoLG of infected mice. Together, our results showed an interplay between the severity of HSK, inflammation of EoLG, and the reduced level of tears after corneal HSV-1 infection.

TFOS DEWS II Tear Film Report.

The members of the Tear Film Subcommittee reviewed the role of the tear film in dry eye disease (DED). The Subcommittee reviewed biophysical and biochemical aspects of tears and how these change in DED. Clinically, DED is characterized by loss of tear volume, more rapid breakup of the tear film and increased evaporation of tears from the ocular surface. The tear film is composed of many substances including lipids, proteins, mucins and electrolytes. All of these contribute to the integrity of the tear film but exactly how they interact is still an area of active research. Tear film osmolarity increases in DED. Changes to other components such as proteins and mucins can be used as biomarkers for DED. The Subcommittee recommended areas for future research to advance our understanding of the tear film and how this changes with DED. The final report was written after review by all Subcommittee members and the entire TFOS DEWS II membership.

Lacritin and other autophagy associated proteins in ocular surface health.

Advantage may be taken of macroautophagy ('autophagy') to promote ocular health. Autophagy continually captures aged or damaged cellular material for lysosomal degradation and recyling. When autophagic flux is chronically elevated, or alternatively deficient, health suffers. Chronic elevation of flux and stress are the consequence of inflammatory cytokines or of dry eye tears but not normal tears invitro. Exogenous tear protein lacritin transiently accelerates flux to restore homeostasis invitro and corneal health invivo, and yet the monomeric active form of lacritin appears to be selectively deficient in dry eye. Tissue transglutaminase-dependent cross-linking of monomer decreases monomer quantity and monomer affinity for coreceptor syndecan-1 thereby abrogating activity. Tissue transglutaminase is elevated in dry eye. Mutation of arylsulfatase A, arylsulfatase B, ceroid-lipofuscinosis neuronal 3, mucolipin, or Niemann-Pick disease type C1 respectively underlie several diseases of apparently insufficient autophagic flux that affect the eye, including: metachromatic leukodystrophy, mucopolysaccharidosis type VI, juvenile-onset Batten disease, mucolipidosis IV, and Niemann-Pick type C associated with myelin sheath destruction of corneal sensory and ciliary nerves and of the optic nerve; corneal clouding, ocular hypertension, glaucoma and optic nerve atrophy; accumulation of 'ceroid-lipofuscin' in surface conjunctival cells, and in ganglion and neuronal cells; decreased visual acuity and retinal dystrophy; and neurodegeneration. For some, enzyme or gene replacement, or substrate reduction, therapy is proving to be successful. Here we discuss examples of restoring ocular surface homeostasis through alteration of autophagy, with particular attention to lacritin.

A thermo-responsive protein treatment for dry eyes.

Millions of Americans suffer from dry eye disease, and there are few effective therapies capable of treating these patients. A decade ago, an abundant protein component of human tears was discovered and named lacritin (Lacrt). Lacrt has prosecretory activity in the lacrimal gland and mitogenic activity at the corneal epithelium. Similar to other proteins placed on the ocular surface, the durability of its effect is limited by rapid tear turnover. Motivated by the rationale that a thermo-responsive coacervate containing Lacrt would have better retention upon administration, we have constructed and tested the activity of a thermo-responsive Lacrt fused to an elastin-like polypeptide (ELP). Inspired from the human tropoelastin protein, ELP protein polymers reversibly phase separate into viscous coacervates above a tunable transition temperature. This fusion construct exhibited the prosecretory function of native Lacrt as illustrated by its ability to stimulate ß-hexosaminidase secretion from primary rabbit lacrimal gland acinar cells. It also increased tear secretion from non-obese diabetic (NOD) mice, a model of autoimmune dacryoadenitis, when administered via intra-lacrimal injection. Lacrt ELP fusion proteins undergo temperature-mediated assembly to form a depot inside the lacrimal gland. We propose that these Lacrt ELP fusion proteins represent a potential therapy for dry eye disease and the strategy of ELP-mediated phase separation may have applicability to other diseases of the ocular surface.

Topical administration of lacritin is a novel therapy for aqueous-deficient dry eye disease.

PURPOSE: Lacritin is a tear glycoprotein with prosecretory, prosurvival, and mitogenic properties. We examined lacritin levels in the tears of Sjögren's syndrome (SS) patients and explored the therapeutic potential of topical lacritin for the treatment of keratoconjunctivitis sicca. METHODS: Tears from healthy controls (n = 14) and SS patients (n = 15) were assayed for lacritin using a C-terminal antibody. In a paired-eye study, autoimmune regulator (Aire) knockout (KO) mice (n = 7) were treated three times daily for 21 days with 10 µL of 4 µM lacritin (left eye) or vehicle (PBS) control (right eye). Tear secretion and ocular surface integrity were assessed at baseline and after treatment. Immunohistochemical staining of CD4+ T cells, cytokeratin-10 (K10), and cytokeratin-12 (K12) expression in the cornea and CD4+ T cell infiltration in the lacrimal glands were assessed. RESULTS: Lacritin monomer (421.8 ± 65.3 ng [SS] vs. 655.8 ± 118.9 ng [controls]; P = 0.05) and C-terminal fragment protein (125 ± 34.1 ng [SS] vs. 399.5 ± 84.3 ng [controls]; P = 0.008) per 100 µL of tear eluate were significantly lower in SS patients. In Aire KO mice treated with lacritin, tear secretion increased by 46% (13.0 ± 3.5 mm vs. 8.9 ± 2.9 mm; P = 0.01) and lissamine green staining score significantly decreased relative to baseline (-0.417 ± 0.06 vs. 0.125 ± 0.07; P = 0.02). Expression of K10 but not K12 in the cornea was significantly decreased in lacritin-treated eyes. Focal CD4+ T cell infiltration of the lacrimal glands was significantly reduced on the lacritin-treated side versus the untreated side. CONCLUSIONS: Lacritin is significantly reduced in the tears of SS patients. Topically administered lacritin has therapeutic potential for the treatment of aqueous-deficient dry eye disease.

A cleavage-potentiated fragment of tear lacritin is bactericidal.

Antimicrobial peptides are important as the first line of innate defense, through their tendency to disrupt bacterial membranes or intracellular pathways and potentially as the next generation of antibiotics. How they protect wet epithelia is not entirely clear, with most individually inactive under physiological conditions and many preferentially targeting Gram-positive bacteria. Tears covering the surface of the eye are bactericidal for Gram-positive and -negative bacteria. Here we narrow much of the bactericidal activity to a latent C-terminal fragment in the prosecretory mitogen lacritin and report that the mechanism combines membrane permeabilization with rapid metabolic changes, including reduced levels of dephosphocoenzyme A, spermidine, putrescine, and phosphatidylethanolamines and elevated alanine, leucine, phenylalanine, tryptophan, proline, glycine, lysine, serine, glutamate, cadaverine, and pyrophosphate. Thus, death by metabolic stress parallels cellular attempts to survive. Cleavage-dependent appearance of the C-terminal cationic amphipathic α-helix is inducible within hours by Staphylococcus epidermidis and slowly by another mechanism, in a chymotrypsin- or leupeptin protease-inhibitable manner. Although bactericidal at low micromolar levels, within a biphasic 1-10 nM dose optimum, the same domain is mitogenic and cytoprotective for epithelia via a syndecan-1 targeting mechanism dependent on heparanase. Thus, the C terminus of lacritin is multifunctional by dose and proteolytic processing and appears to play a key role in the innate protection of the eye, with wider potential benefit elsewhere as lacritin flows from exocrine secretory cells.

Cytoprotective effect of lacritin on human corneal epithelial cells exposed to benzalkonium chloride in vitro.

PURPOSE: Benzalkonium chloride (BAK) is the most commonly found preservative in eye drops, and has been shown to cause ocular surface inflammation and toxicity. Lacritin is a human tear glycoprotein secreted from the lacrimal glands that has been found to be cytoprotective. This study was designed to determine if the presence of lacritin confers protection to a cultured human corneal epithelial (HCE) cell line, CRL-11515, and primary HCE cells after exposure to the ocular preservative agent BAK. MATERIALS AND METHODS: Recombinant human lacritin was cloned into intein fusion vectors, expressed in E. coli, and purified on chitin beads and DEAE Sepharose. Metabolic curves were established using the MTT assay after exposure of sub-confluent CRL-11515 cells to BAK or lacritin. Western blot analysis of lipidated LC3 (LC3-II) provided a measure of autophagy in CRL-11515 cells exposed to lacritin and/or BAK. RESULTS: BAK reduced CRL-11515 cellular metabolic activity in a time- and dose-dependent manner. BAK-induced cellular stress was evident by elevated autophagy that increased with rising concentrations of BAK compared to control (p < 0.05). Lacritin increased HCE cell proliferation at an optimal dose of 1 nM. Preconditioning HCE cells with 1 nM lacritin for 24 h prior to BAK exposure significantly dampened levels of LC3-II (p < 0.05) and promoted a significant increase in cellular metabolic activity (p < 0.01) compared to BAK alone. CONCLUSIONS: These results suggest lacritin protects cultured HCE cells stressed with BAK. Lacritin may have the potential to be used as a topical adjunctive therapy in eyes chronically exposed to BAK.

Lacritin and the tear proteome as natural replacement therapy for dry eye.

Tear proteins are potential biomarkers, drug targets, and even biotherapeutics. As a biotherapeutic, a recombinant tear protein might physiologically rescue the ocular surface when a deficiency is detected. Such a strategy pays more attention to the natural prosecretory and protective properties of the tear film and seeks to alleviate symptoms by addressing cause, rather than the current palliative, non-specific and temporary approaches. Only a handful of tear proteins appear to be selectively downregulated in dry eye, the most common eye disease. Lacritin and lipocalin-1 are two tear proteins selectively deficient in dry eye. Both proteins influence ocular surface health. Lacritin is a prosecretory mitogen that promotes basal tearing when applied topically. Levels of active monomeric lacritin are negatively regulated by tear tissue transglutaminase, whose expression is elevated in dry eye with ocular surface inflammation. Lipocalin-1 is the master lipid sponge of the ocular surface, without which residual lipids could interfere with epithelial wetting. It also is a carrier for vitamins and steroid hormones, and is a key endonuclease. Accumulation of DNA in tears is thought to be proinflammatory. Functions of these and other tear proteins may be influenced by protein-protein interactions. Here we discuss new advances in lacritin biology and provide an overview on lipocalin-1, and newly identified members of the tear proteome.

Lacritin rescues stressed epithelia via rapid forkhead box O3 (FOXO3)-associated autophagy that restores metabolism.

Homeostasis is essential for cell survival. However, homeostatic regulation of surface epithelia is poorly understood. The eye surface, lacking the cornified barrier of skin, provides an excellent model. Tears cover the surface of the eye and are deficient in dry eye, the most common eye disease affecting at least 5% of the world's population. Only a tiny fraction of the tear proteome appears to be affected, including lacritin, an epithelium-selective mitogen that promotes basal tearing when topically applied to rabbit eyes. Here we show that homeostasis of cultured corneal epithelia is entirely lacritin-dependent and elucidate the mechanism as a rapid autophagic flux to promptly restore cellular metabolism and mitochondrial fusion in keeping with the short residence time of lacritin on the eye. Accelerated flux appears to be derived from lacritin-stimulated acetylation of FOXO3 as a novel ligand for ATG101 and coupling of stress-acetylated FOXO1 with ATG7 (which remains uncoupled without lacritin) and be sufficient to selectively divert huntingtin mutant Htt103Q aggregates largely without affecting non-aggregated Htt25Q. This is in keeping with stress as a prerequisite for lacritin-stimulated autophagy. Lacritin targets the cell surface proteoglycan syndecan-1 via its C-terminal amino acids Leu(108)-Leu(109)-Phe(112) and is also available in saliva, plasma, and lung lavage. Thus, lacritin may promote epithelial homeostasis widely.

Targeting of heparanase-modified syndecan-1 by prosecretory mitogen lacritin requires conserved core GAGAL plus heparan and chondroitin sulfate as a novel hybrid binding site that enhances selectivity.

Cell surface heparan sulfate (HS) proteoglycans shape organogenesis and homeostasis by capture and release of morphogens through mechanisms largely thought to exclude the core protein domain. Nevertheless, heparanase deglycanation of the N-terminal HS-rich domain of syndecan-1 (SDC1), but not SDC2 or -4, is a prerequisite for binding of the prosecretory mitogen lacritin (Ma, P., Beck, S. L., Raab, R. W., McKown, R. L., Coffman, G. L., Utani, A., Chirico, W. J., Rapraeger, A. C., and Laurie, G. W. (2006) Heparanase deglycanation of syndecan-1 is required for binding of the epithelial-restricted prosecretory mitogen lacritin. J. Cell Biol. 174, 1097-1106). We now report that the conserved and hydrophobic GAGAL domain in SDC1, adjacent to predicted HS substitution sites, is necessary to ligate and substantially enhance the α-helicity of the amphipathic C terminus of lacritin. Swapping out GAGAL for GADED in SDC2 or for GDLDD in SDC4 (both less hydrophobic) abrogated binding. HS and chondroitin sulfate are also essential. Both are detected in the N terminus, and when incubated with antibodies HS4C3 (anti-HS) or IO3H10 (anti-chondroitin sulfate), binding was absent, as occurred when all three N-terminal glycosaminoglycan substitution sites were mutated to alanine or when cells were treated with 4-methylumbelliferyl-ß-d-xylopyranoside or chlorate to suppress glycosaminoglycan substitution or sulfation, respectively. SDC1 interacts with the hydrophobic face of lacritin via Leu-108/Leu-109/Phe-112 as well as with Glu-103/Lys-107 and Lys-111 of the largely cationic face. Carving a hybrid hydrophobic/electrostatic docking site out of SDC1 in a manner dependent on endogenous heparanase is a dynamic process appropriate for subtle or broad epithelial regulation in morphogenesis, health, and disease.